Low-Level Pilin Expression Allows for Substantial DNA Transformation Competence in Neisseria gonorrhoeae

ABSTRACT The gonococcal pilus is a major virulence factor that has well-established roles in mediating epithelial cell adherence and DNA transformation. Gonococci expressing four gonococcal pilin variants with distinct piliation properties under control of the lac regulatory system were grown in different levels of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). These pilin variants expressed various levels of pilin message and pilin protein in response to the level of IPTG in the growth medium. Moreover, posttranslational modifications of the variant pilin proteins were detected, including S-pilin production and glycosylation. The ratio of the modified and unmodified pilin forms did not substantially change with different levels of pilin expression, showing that these modifications are not linked to pilin expression levels. DNA transformation competence was also influenced by IPTG levels in the growth medium. Substantial increases in transformation competence over an isogenic, nonpiliated mutant were observed when limited amounts of three of the pilin variants were expressed. Immunoelectron microscopy showed that when limited amounts of pilin are expressed, pili are rare and do not explain the pilin-dependent transformation competence. This pilin-dependent transformation competence required prepilin processing, the outer membrane secretin PilQ, and the twitching-motility-regulating protein PilT. These requirements show that a fully functional pilus assembly apparatus is required for DNA uptake when limited pilin is produced. We conclude that the pilus assembly apparatus functions to import DNA into the bacterial cell in a pilin-dependent manner but that extended pili are not required for transformation competence.

Download Full-text

Natural Competence for DNA Transformation by Legionella pneumophila and Its Association with Expression of Type IV Pili

Journal of Bacteriology ◽

10.1128/jb.181.5.1395-1402.1999 ◽

1999 ◽

Vol 181 (5) ◽

pp. 1395-1402 ◽

Cited By ~ 142

Author(s):

Barbara J. Stone ◽

Yousef Abu Kwaik

Keyword(s):

Legionella Pneumophila ◽

Plasmid Dna ◽

Mammalian Cells ◽

Type Iv Pili ◽

Dna Transformation ◽

Dna Uptake ◽

Chromosomal Dna ◽

Type Iv ◽

Type Iv Pilin ◽

Pilin Protein

ABSTRACT We have recently described the expression of two pili of different lengths on the surface of Legionella pneumophila (B. J. Stone and Y. Abu Kwaik, Infect. Immun. 66:1768–1775, 1998). Production of long pili requires a functional pilE L locus, encoding a type IV pilin protein. Since type IV pili in Neisseria gonorrhoeaeare associated with competence for DNA transformation, we examined the competence of L. pneumophila for DNA transformation under conditions that allowed the expression of type IV pili. We show that L. pneumophila is naturally competent for DNA transformation by isogenic chromosomal DNA and by plasmid DNA containing L. pneumophila DNA. Many different L. pneumophila loci are able to transform L. pneumophilaafter addition of plasmid DNA, including gspA,ppa, asd, and pilE L. The transformation frequency is reduced when competing DNA containing either L. pneumophila DNA or vector sequences is added to the bacteria, suggesting that uptake-specific sequences may not be involved in DNA uptake. Competence for DNA transformation correlates with expression of the type IV pili, and apilE L mutant defective in expression of type IV pili is not competent for DNA transformation. Complementation of the mutant for competence is restored by the reintroduction of a cosmid that restores production of type IV pili. Minimal competence is restored to the mutant by introduction of pilE Lalone. We conclude that competence for DNA transformation in L. pneumophila is associated with expression of the type IV pilus and results in recombination of L. pneumophila DNA into the chromosome. Since expression of type IV pili also facilitates attachment of L. pneumophila to mammalian cells and protozoa, we designated the type IV pili CAP (for competence- and adherence-associated pili).

Download Full-text

Regulation of igaA and the Rcs System by the MviA Response Regulator in Salmonella enterica

Journal of Bacteriology ◽

10.1128/jb.01519-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2743-2752 ◽

Cited By ~ 6

Author(s):

Clara B. García-Calderón ◽

Josep Casadesús ◽

Francisco Ramos-Morales

Keyword(s):

Membrane Protein ◽

Salmonella Enterica ◽

Response Regulator ◽

Regulatory System ◽

Enteric Bacteria ◽

Lon Protease ◽

Dependent Manner ◽

Independent Manner ◽

Serovar Typhimurium

ABSTRACT IgaA is a membrane protein that prevents overactivation of the Rcs regulatory system in enteric bacteria. Here we provide evidence that igaA is the first gene in a σ70-dependent operon of Salmonella enterica serovar Typhimurium that also includes yrfG, yrfH, and yrfI. We also show that the Lon protease and the MviA response regulator participate in regulation of the igaA operon. Our results indicate that MviA regulates igaA transcription in an RpoS-dependent manner, but the results also suggest that MviA may regulate RcsB activation in an RpoS- and IgaA-independent manner.

Download Full-text

PathwayMatcher: proteoform-centric network construction enables fine-granularity multiomics pathway mapping

GigaScience ◽

10.1093/gigascience/giz088 ◽

2019 ◽

Vol 8 (8) ◽

Author(s):

Luis Francisco Hernández Sánchez ◽

Bram Burger ◽

Carlos Horro ◽

Antonio Fabregat ◽

Stefan Johansson ◽

...

Keyword(s):

Protein Interactions ◽

Posttranslational Modifications ◽

Knowledge Bases ◽

Protein Isoforms ◽

Biomedical Data ◽

Protein Protein Interactions ◽

Gene Sets ◽

Functional Knowledge ◽

Pathway Analyses ◽

Different Levels

Abstract Background Mapping biomedical data to functional knowledge is an essential task in bioinformatics and can be achieved by querying identifiers (e.g., gene sets) in pathway knowledge bases. However, the isoform and posttranslational modification states of proteins are lost when converting input and pathways into gene-centric lists. Findings Based on the Reactome knowledge base, we built a network of protein-protein interactions accounting for the documented isoform and modification statuses of proteins. We then implemented a command line application called PathwayMatcher (github.com/PathwayAnalysisPlatform/PathwayMatcher) to query this network. PathwayMatcher supports multiple types of omics data as input and outputs the possibly affected biochemical reactions, subnetworks, and pathways. Conclusions PathwayMatcher enables refining the network representation of pathways by including proteoforms defined as protein isoforms with posttranslational modifications. The specificity of pathway analyses is hence adapted to different levels of granularity, and it becomes possible to distinguish interactions between different forms of the same protein.

Download Full-text

SseL Is a Salmonella-Specific Translocated Effector Integrated into the SsrB-Controlled Salmonella Pathogenicity Island 2 Type III Secretion System

Infection and Immunity ◽

10.1128/iai.00985-06 ◽

2006 ◽

Vol 75 (2) ◽

pp. 574-580 ◽

Cited By ~ 48

Author(s):

Brian K. Coombes ◽

Michael J. Lowden ◽

Jennifer L. Bishop ◽

Mark E. Wickham ◽

Nat F. Brown ◽

...

Keyword(s):

Virulence Factors ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Regulatory System ◽

Pathogenicity Island ◽

Secretion System ◽

Host Cells ◽

Dependent Manner ◽

Host Colonization ◽

Type Iii

ABSTRACT Bacterial pathogens use horizontal gene transfer to acquire virulence factors that influence host colonization, alter virulence traits, and ultimately shape the outcome of disease following infection. One hallmark of the host-pathogen interaction is the prokaryotic type III secretion system that translocates virulence factors into host cells during infection. Salmonella enterica possesses two type III secretion systems that are utilized during host colonization and intracellular replication. Salmonella pathogenicity island 2 (SPI2) is a genomic island containing approximately 30 contiguous genes required to assemble a functional secretion system including the two-component regulatory system called SsrA-SsrB that positively regulates transcription of the secretion apparatus. We used transcriptional profiling with DNA microarrays to search for genes that coregulate with the SPI2 type III secretion machinery in an SsrB-dependent manner. Here we report the identification of a Salmonella-specific translocated effector called SseL that is required for full virulence during murine typhoid-like disease. Analysis of infected macrophages using fluorescence-activated cell sorting revealed that sseL is induced inside cells and requires SsrB for expression. SseL is retained predominantly in the cytoplasm of infected cells following translocation by the type III system encoded in SPI2. Animal infection experiments with sseL mutant bacteria indicate that integration of SseL into the SsrB response regulatory system contributes to systemic virulence of this pathogen.

Download Full-text

Energy-Generating Enzymes of Burkholderia cepacia and Their Interactions with Macrophages

Journal of Bacteriology ◽

10.1128/jb.185.10.3167-3178.2003 ◽

2003 ◽

Vol 185 (10) ◽

pp. 3167-3178 ◽

Cited By ~ 10

Author(s):

Vasu Punj ◽

Rachna Sharma ◽

Olga Zaborina ◽

A. M. Chakrabarty

Keyword(s):

Cystic Fibrosis ◽

Cell Death ◽

Growth Medium ◽

Burkholderia Cepacia ◽

Dependent Manner ◽

Bacterial Protein ◽

Sepharose Column ◽

Cellular Energetics ◽

Cystic Fibrosis Isolate ◽

High Cytotoxicity

ABSTRACT We previously demonstrated that several clinical and environmental isolates of Burkholderia cepacia secreted ATP-utilizing enzymes to the medium; the secretion of these enzymes by cystic fibrosis lung isolate strain 38 was shown to be greatly enhanced in the presence of α2-macroglobulin. Fractionation of the growth medium of cystic fibrosis isolate strain 71 belonging to genomovar I demonstrated the presence of two additional proteins, homologues of Pseudomonas aeruginosa azurin and cytochrome c 551, which are normally involved in electron transfer during denitrification. A Q-Sepharose column flowthrough fraction of the growth medium of B. cepacia strain 71 enriched with the azurin and cytochrome c 551 homologues triggered apoptosis in macrophages and mast cells, leading to their death. Incubation of the Q-Sepharose column flowthrough fraction with antiazurin and anti-cytochrome c 551 antibodies greatly reduced cell death. We cloned and hyperexpressed a gene from B. cepacia strain 71 that encodes the homologue of P. aeruginosa azurin. Such azurin homologues were detected in the growth medium of several strains belonging to genomovars I, III, and VI but not in the growth medium of strains belonging to other genomovars. The growth medium of the strains that elaborated the azurin homologue had high cytotoxicity towards macrophages. Purified azurin homologue was shown to induce apoptosis in macrophages in a caspase-dependent manner and was localized in both the cytosol and nucleus when incubated with or microinjected into macrophages. This is an interesting example of the interaction of a bacterial protein normally involved in cellular energetics with macrophages to effect their cell death.

Download Full-text

A Novel Ex Vivo Model of Aortic Valve Calcification. A Preliminary Report

Frontiers in Pharmacology ◽

10.3389/fphar.2020.568764 ◽

2020 ◽

Vol 11 ◽

Author(s):

Arsenii Zabirnyk ◽

Maria del Mar Perez ◽

Marc Blasco ◽

Kåre-Olav Stensløkken ◽

Miguel D. Ferrer ◽

...

Keyword(s):

Aortic Valve ◽

Growth Medium ◽

Ex Vivo ◽

Growth Media ◽

Dependent Manner ◽

Ex Vivo Model ◽

Aortic Valves ◽

Valve Calcification ◽

Calcium Accumulation ◽

Aortic Valve Leaflets

Background: No pharmacological treatment exists to prevent or stop the calcification process of aortic valves causing aortic stenosis. The aim of this study was to develop a robust model of induced calcification in whole aortic valve leaflets which could be suitable for studies of the basic mechanisms and for testing potentially inhibitory drugs.Methods: Pig hearts were obtained from a commercial abattoir. The aortic valve leaflets were dissected free and randomized between experimental groups. Whole leaflets were cultured in individual wells. Two growth media were used for cultivation: standard growth medium and an antimyofibroblastic growth medium. The latter was employed to inhibit contraction of the leaflet into a ball-like structure. Calcification was induced in the growth medium by supplementation with an osteogenic medium. Leaflets were cultivated for four weeks and medium was changed every third day. To block calcification, the inhibitor SNF472 (a formulation of the hexasodium salt of myo-inositol hexaphosphate hexasodium salt) was used at concentrations between 1 and 100 µM. After cultivation for four weeks the leaflets were snap frozen in liquid nitrogen and kept at −80 °C until blind assessment of the calcium amount in leaflets by inductively coupled plasma optical emission spectroscopy. For statistical analysis, a Kruskal–Wallis test with Dunn’s post-test was applied.Results: Osteodifferentiation with calcium accumulation was in principle absent when standard medium was used. However, when the antimyofibroblastic medium was used, a strong calcium accumulation was induced (p = 0.006 compared to controls), and this was blocked in a dose-dependent manner by the calcification inhibitor SNF472 (p = 0.008), with an EC50 of 3.3 µM.Conclusion: A model of experimentally induced calcification in cultured whole leaflets from porcine aortic valves was developed. This model can be useful for studying the basic mechanisms of valve calcification and to test pharmacological approaches to inhibit calcification.

Download Full-text

Pseudomonas aeruginosatype IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity

10.1101/192062 ◽

2017 ◽

Author(s):

Victoria A. Marko ◽

Sara L.N. Kilmury ◽

Lesley T. MacNeil ◽

Lori L. Burrows

Keyword(s):

Pseudomonas Aeruginosa ◽

Virulence Factors ◽

Feedback Inhibition ◽

Regulatory System ◽

Type Iv Pili ◽

Infection Model ◽

Type Iv ◽

Wide Range ◽

Lung Infections ◽

Pilus Assembly

AbstractType IV pili are expressed by a wide range of prokaryotes, including the opportunistic pathogenPseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The pilus is composed mainly of major pilin subunits while the low abundance minor pilins FimU-PilVWXE and the putative adhesin PilY1 prime pilus assembly and are proposed to form the pilus tip. The minor pilins and PilY1 are encoded in an operon that is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 is proposed to be a mechanosensory component that - in conjunction with minor pilins - triggers up-regulation of acute virulence phenotypes upon surface attachment. Here, we investigated the link between the minor pilins and virulence.pilW, pilX, andpilY1mutants had reduced virulence towardsCaenorhabditis elegansrelative to wild type or a major pilin mutant, implying a role in pathogenicity that is independent of pilus assembly. We hypothesized that loss of specific minor pilins relieves feedback inhibition on FimS-AlgR, increasing transcription of the minor pilin operon and other members of the AlgR regulon. Reporter assays confirmed that FimS-AlgR were required for the increased expression from the minor pilin operon promoter upon loss of select minor pilins. Overexpression of AlgR or its hyperactivation via point mutation reduced virulence, and the virulence defects ofpilW,pilX, andpilY1mutants were dependent on FimS-AlgR expression and activation. We propose that PilY1 and the minor pilins inhibit their own expression, and that loss of these proteins leads to FimS-mediated activation of AlgR and reduced expression of acute-phase virulence factors. This mechanism could contribute to adaptation ofP. aeruginosain chronic lung infections, as mutations in the minor pilin operon result in the loss of piliation and increased expression of AlgR-dependent virulence factors – such as alginate – that are characteristic of such infections.Author summaryPseudomonas aeruginosacauses dangerous infections, including chronic lung infections in cystic fibrosis patients. It uses many strategies to infect its hosts, including deployment of grappling hook-like fibres called type IV pili. Among the components involved in assembly and function of the pilus are five proteins called minor pilins that - along with a larger protein called PilY1 - may help the pilus attach to surfaces. In a roundworm infection model, loss of PilY1 and specific minor pilins delayed killing, while loss of other pilus proteins did not. We traced this effect to increased activation of the FimS-AlgR regulatory system that inhibits expression of virulence factors used to initiate infections, while positively regulating chronic infection traits such as alginate production, a phenotype called mucoidy. A disruption in the appropriate timing of FimS-AlgR-dependent virulence factor expression when select minor pilins or PilY1 are missing may explain why those pilus-deficient mutants have reduced virulence compared with others whose products are not under FimS-AlgR control. Increased FimS-AlgR activity upon loss of PilY1 and specific minor pilins could help to explain the frequent co-occurrence of the non-piliated and mucoid phenotypes that are hallmarks of chronicP. aeruginosalung infections.

Download Full-text

E3 ligase MDM2 mediates urea transporter-A1 ubiquitination under either constitutive or stimulatory conditions

AJP Renal Physiology ◽

10.1152/ajprenal.00316.2019 ◽

2019 ◽

Vol 317 (5) ◽

pp. F1331-F1341

Author(s):

Hua Su ◽

Chen Ye ◽

Jeff M. Sands ◽

Chun Zhang

Keyword(s):

Posttranslational Modifications ◽

Binding Capacity ◽

Diabetic Rat ◽

Dependent Manner ◽

Urea Transporter ◽

Subcellular Trafficking ◽

Complex Issue ◽

Murine Double Minute ◽

The Stability

Posttranslational modifications are essential for the regulation of urea transporter-A1 (UT-A1), among which ubiquitination is a rather attractive and complex issue. Previously, our group reported that murine double minute 2 (MDM2) is one of the E3 ubiquitin ligases for UT-A1, and, later, we showed that ubiquitination contributes to the subcellular trafficking and stability of UT-A1. In the present study, we discovered that MDM2 interacts with UT-A1 in an AP50 (a component of the clathrin-coated pit)-dependent manner. However, their binding is irrelevant to the phosphorylatory status of UT-A1. Next, our findings indicated that MDM2 decreases the stability of either total or membrane UT-A1. On the cell membrane, MDM2 and ubiquitinated UT-A1 are both distributed in the lipid raft domain, and their linkage is obviously enhanced under forskolin (FSK) stimulation. In line with these results, in the diabetic rat, not only MDM2 but also ubiquitinated UT-A1 are intensified. Also, in vitro high glucose and angiotensin II play similar roles as FSK does on the association of MDM2 with UT-A1. In conclusion, MDM2 binds with UT-A1 and mediates its ubiquitination and degradation in an AP50-dependent manner, and their binding capacity is strengthened under FSK and diabetic milieu.

Download Full-text

Natural DNA Transformation Is Functional in Lactococcus lactis subsp. cremoris KW2

Applied and Environmental Microbiology ◽

10.1128/aem.01074-17 ◽

2017 ◽

Vol 83 (16) ◽

Cited By ~ 5

Author(s):

Blandine David ◽

Amandine Radziejwoski ◽

Frédéric Toussaint ◽

Laetitia Fontaine ◽

Marie Henry de Frahan ◽

...

Keyword(s):

Lactic Acid ◽

Lactococcus Lactis ◽

Natural Transformation ◽

Single Point ◽

Adaptor Protein ◽

Single Point Mutation ◽

Dna Transformation ◽

Dna Uptake ◽

Genetic Traits ◽

Content Type

ABSTRACT Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species. IMPORTANCE Lactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.

Download Full-text

Carbon Sources for Yeast Growth as a Precondition of Hydrogen Peroxide Induced Hormetic Phenotype

International Journal of Microbiology ◽

10.1155/2015/697813 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 11

Author(s):

Ruslana Vasylkovska ◽

Natalia Petriv ◽

Halyna Semchyshyn

Keyword(s):

Hydrogen Peroxide ◽

Growth Medium ◽

Carbon Sources ◽

Carbon Substrate ◽

Dependent Manner ◽

Yeast Growth ◽

Concentration Dependent Manner ◽

Reproductive Ability ◽

The Relationship ◽

Hormetic Response

Hormesis is a phenomenon of particular interest in biology, medicine, pharmacology, and toxicology. In this study, we investigated the relationship between H2O2-induced hormetic response inS. cerevisiaeand carbon sources in yeast growth medium. In general, our data indicate that (i) hydrogen peroxide induces hormesis in a concentration-dependent manner; (ii) the effect of hydrogen peroxide on yeast reproductive ability depends on the type of carbon substrate in growth medium; and (iii) metabolic and growth rates as well as catalase activity play an important role in H2O2-induced hormetic response in yeast.

Download Full-text