scholarly journals The Bacteroides thetaiotaomicron Protein Bacteroides Host Factor A Participates in Integration of the Integrative Conjugative Element CTnDOT into the Chromosome

2015 ◽  
Vol 197 (8) ◽  
pp. 1339-1349 ◽  
Author(s):  
Kenneth Ringwald ◽  
Jeffrey Gardner
Keyword(s):  
Antibiotic Resistance Genes ◽  
Host Factor ◽  
Host Cells ◽  
Recombination Reaction ◽  
Host Chromosome ◽  
Content Type ◽  
Integrative And Conjugative Elements ◽  
Integrative Conjugative Element ◽  
Associated Proteins ◽  
A Site

ABSTRACTCTnDOT is a conjugative transposon found inBacteroidesspecies. It encodes multiple antibiotic resistances and is stimulated to transfer by exposure to tetracycline. CTnDOT integration into the host chromosome requires IntDOT and a previously unknown host factor. We have identified a protein, designated BHFa (Bacteroideshost factor A), that participates in integrative recombination. BHFa is the first host factor identified for a site-specific recombination reaction in the CTnDOT family of integrative and conjugative elements. Based on the amino acid sequence of BHFa, the ability to bind specifically to 4 sites in theattDOTDNA, and its activity in the integration reaction, BHFa is a member of the IHF/HU family of nucleoid-associated proteins. Other DNA bending proteins that bind DNA nonspecifically can substitute for BHFa in the integration reaction.IMPORTANCEBacteroidesspecies are normal members of the human colonic microbiota. These species can harbor and spread self-transmissible genetic elements (integrative conjugative elements [ICEs]) that contain antibiotic resistance genes. This work describes the role of a protein, BHFa, and its importance in the integration reaction required for the element CTnDOT to persist inBacteroideshost cells.

scholarly journals Immunomodulatory Effects of Pneumococcal Extracellular Vesicles on Cellular and Humoral Host Defenses

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Mario Codemo ◽  
Sandra Muschiol ◽  
Federico Iovino ◽  
Priyanka Nannapaneni ◽  
Laura Plant ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Plasma Membrane ◽  
Streptococcus Pneumoniae ◽  
Extracellular Vesicles ◽  
Factor H ◽  
Host Cells ◽  
Immunomodulatory Effects ◽  
Content Type ◽  
Associated Proteins ◽  
Tract Infections

ABSTRACTGram-positive bacteria, including the major respiratory pathogenStreptococcus pneumoniae, were recently shown to produce extracellular vesicles (EVs) that likely originate from the plasma membrane and are released into the extracellular environment. EVs may function as cargo for many bacterial proteins, however, their involvement in cellular processes and their interactions with the innate immune system are poorly understood. Here, EVs from pneumococci were characterized and their immunomodulatory effects investigated. Pneumococcal EVs were protruding from the bacterial surface and released into the medium as 25 to 250 nm lipid stained vesicles containing a large number of cytosolic, membrane, and surface-associated proteins. The cytosolic pore-forming toxin pneumolysin was significantly enriched in EVs compared to a total bacterial lysate but was not required for EV formation. Pneumococcal EVs were internalized into A549 lung epithelial cells and human monocyte-derived dendritic cells and induced proinflammatory cytokine responses irrespective of pneumolysin content. EVs from encapsulated pneumococci were recognized by serum proteins, resulting in C3b deposition and formation of C5b-9 membrane attack complexes as well as factor H recruitment, depending on the presence of the choline binding protein PspC. Addition of EVs to human serum decreased opsonophagocytic killing of encapsulated pneumococci. Our data suggest that EVs may act in an immunomodulatory manner by allowing delivery of vesicle-associated proteins and other macromolecules into host cells. In addition, EVs expose targets for complement factors in serum, promoting pneumococcal evasion of humoral host defense.IMPORTANCEStreptococcus pneumoniaeis a major contributor to morbidity and mortality worldwide, being the major cause of milder respiratory tract infections such as otitis and sinusitis and of severe infections such as community-acquired pneumonia, with or without septicemia, and meningitis. More knowledge is needed on how pneumococci interact with the host, deliver virulence factors, and activate immune defenses. Here we show that pneumococci form extracellular vesicles that emanate from the plasma membrane and contain virulence properties, including enrichment of pneumolysin. We found that pneumococcal vesicles can be internalized into epithelial and dendritic cells and bind complement proteins, thereby promoting pneumococcal evasion of complement-mediated opsonophagocytosis. They also induce pneumolysin-independent proinflammatory responses. We suggest that these vesicles can function as a mechanism for delivery of pneumococcal proteins and other immunomodulatory components into host cells and help pneumococci to avoid complement deposition and phagocytosis-mediated killing, thereby possibly contributing to the symptoms found in pneumococcal infections.

scholarly journals Investigating the Relatedness of Enteroinvasive Escherichia coli to Other E. coli and Shigella Isolates by Using Comparative Genomics

2016 ◽  
Vol 84 (8) ◽  
pp. 2362-2371 ◽  
Author(s):  
Tracy H. Hazen ◽  
Susan R. Leonard ◽  
Keith A. Lampel ◽  
David W. Lacher ◽  
Anthony T. Maurelli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genomic Analysis ◽  
Host Cells ◽  
Comparative Genomic ◽  
Virulence Plasmid ◽  
Phylogenomic Analysis ◽  
Content Type ◽  
E Coli ◽  
Associated Proteins ◽  
Human Illness

EnteroinvasiveEscherichia coli(EIEC) is a unique pathovar that has a pathogenic mechanism nearly indistinguishable from that ofShigellaspecies. In contrast to isolates of the fourShigellaspecies, which are widespread and can be frequent causes of human illness, EIEC causes far fewer reported illnesses each year. In this study, we analyzed the genome sequences of 20 EIEC isolates, including 14 first described in this study. Phylogenomic analysis of the EIEC genomes demonstrated that 17 of the isolates are present in three distinct lineages that contained only EIEC genomes, compared to reference genomes from each of theE. colipathovars andShigellaspecies. Comparative genomic analysis identified genes that were unique to each of the three identified EIEC lineages. While many of the EIEC lineage-specific genes have unknown functions, those with predicted functions included a colicin and putative proteins involved in transcriptional regulation or carbohydrate metabolism.In silicodetection of theShigellavirulence plasmid (pINV), which is essential for the invasion of host cells, demonstrated that a form of pINV was present in nearly all EIEC genomes, but the Mxi-Spa-Ipa region of the plasmid that encodes the invasion-associated proteins was absent from several of the EIEC isolates. The comparative genomic findings in this study support the hypothesis that multiple EIEC lineages have evolved independently from multiple distinct lineages ofE. colivia the acquisition of theShigellavirulence plasmid and, in some cases, theShigellapathogenicity islands.

scholarly journals Effects of Three Different Nucleoid-Associated Proteins Encoded on IncP-7 Plasmid pCAR1 on Host Pseudomonas putida KT2440

2015 ◽  
Vol 81 (8) ◽  
pp. 2869-2880 ◽  
Author(s):  
Chiho Suzuki-Minakuchi ◽  
Ryusuke Hirotani ◽  
Masaki Shintani ◽  
Toshiharu Takeda ◽  
Yurika Takahashi ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Structural Instability ◽  
Metabolic Phenotype ◽  
Pseudomonas Putida Kt2440 ◽  
Host Chromosome ◽  
Content Type ◽  
Inorganic Ion ◽  
Segregational Stability ◽  
Genes Encoding ◽  
Associated Proteins

ABSTRACTNucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption ofpmr,pnd, andphuwere assessed in hostPseudomonas putidaKT2440. Whenpmrandpndorpmrandphuwere simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation ofpmr, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype.

scholarly journals Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Gurunathan Laxmikanthan ◽  
Chen Xu ◽  
Axel F Brilot ◽  
David Warren ◽  
Lindsay Steele ◽  
...  
Keyword(s):  
Dna Recombination ◽  
Structural Complexity ◽  
Holliday Junction ◽  
Structural Basis ◽  
Recombination Reaction ◽  
Host Chromosome ◽  
Protein Subunits ◽  
E Coli ◽  
Molecular Machinery ◽  
A Site

The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural complexity. We report here the structure of the intact functional assembly responsible for regulating and executing a site-specific DNA recombination reaction. The assembly is a 240-bp Holliday junction (HJ) bound specifically by 11 protein subunits. This higher-order complex is a key intermediate in the tightly regulated pathway for the excision of bacteriophage λ viral DNA out of the E. coli host chromosome, an extensively studied paradigmatic model system for the regulated rearrangement of DNA. Our results provide a structural basis for pre-existing data describing the excisive and integrative recombination pathways, and they help explain their regulation.

scholarly journals Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments

2014 ◽  
Vol 80 (12) ◽  
pp. 3597-3603 ◽  
Author(s):  
Vicki Adams ◽  
Radhika Bantwal ◽  
Lauren Stevenson ◽  
Jackie K. Cheung ◽  
Milena M. Awad ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Genetic Manipulation ◽  
Antibiotic Resistance Genes ◽  
Dna Fragments ◽  
Site Specific ◽  
Content Type ◽  
E Coli ◽  
Marker Recycling ◽  
A Site

ABSTRACTTnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451and Tn4453inClostridium perfringensandClostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids inEscherichia coliand from marked chromosomalC. perfringensmutants. This methodology enabled the construction of aC. perfringensplc virRdouble mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic toE. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes inE. colifor subsequent expression in the heterologous hostC. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.

scholarly journals Identification and Characterization of Outer Membrane Vesicle-Associated Proteins in Salmonella enterica Serovar Typhimurium

2014 ◽  
Vol 82 (10) ◽  
pp. 4001-4010 ◽  
Author(s):  
Jaewoo Bai ◽  
Seul I Kim ◽  
Sangryeol Ryu ◽  
Hyunjin Yoon
Keyword(s):  
Outer Membrane ◽  
Virulence Factors ◽  
Salmonella Enterica ◽  
Minimal Medium ◽  
Environmental Changes ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Associated Proteins

ABSTRACTSalmonella entericaserovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enableSalmonellato survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influencedSalmonellasurvival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into howSalmonellautilizes OMVs to adapt to environmental changes.

scholarly journals The Integrative Conjugative Element (ICE) ofMycoplasma agalactiae: Key Elements Involved in Horizontal Dissemination and Influence of Coresident ICEs

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Eric Baranowski ◽  
Emilie Dordet-Frisoni ◽  
Eveline Sagné ◽  
Marie-Claude Hygonenq ◽  
Gabriela Pretre ◽  
...  
Keyword(s):  
Cooperative Behavior ◽  
Insertion Site ◽  
Diverse Group ◽  
Closely Related Species ◽  
Host Chromosome ◽  
Content Type ◽  
Mycoplasma Agalactiae ◽  
Complex Interactions ◽  
Integrative Conjugative Element ◽  
Integrative Conjugative Elements

ABSTRACTThe discovery of integrative conjugative elements (ICEs) in wall-less mycoplasmas and the demonstration of their role in massive gene flows within and across species have shed new light on the evolution of these minimal bacteria. Of these, the ICE of the ruminant pathogenMycoplasma agalactiae(ICEA) represents a prototype and belongs to a new clade of the Mutator-like superfamily that has no preferential insertion site and often occurs as multiple chromosomal copies. Here, functional genomics and mating experiments were combined to address ICEA functions and define the minimal ICEA chassis conferring conjugative properties toM. agalactiae. Data further indicated a complex interaction among coresident ICEAs, since the minimal ICEA structure was influenced by the occurrence of additional ICEA copies that cantrans-complement conjugation-deficient ICEAs. However, this cooperative behavior was limited to the CDS14 surface lipoprotein, which is constitutively expressed by coresident ICEAs, and did not extend to other ICEA proteins, including thecis-acting DDE recombinase and components of the mating channel whose expression was detected only sporadically. Remarkably, conjugation-deficient mutants containing a single ICEA copy knocked out incds14can be complemented by neighboring cells expressing CDS14. This result, together with those revealing the conservation of CDS14 functions in closely related species, may suggest a way for mycoplasma ICEs to extend their interaction outside their chromosomal environment. Overall, this report provides a first model of conjugative transfer in mycoplasmas and offers valuable insights into understanding horizontal gene transfer in this highly adaptive and diverse group of minimal bacteria.IMPORTANCEIntegrative conjugative elements (ICEs) are self-transmissible mobile genetic elements that are key mediators of horizontal gene flow in bacteria. Recently, a new category of ICEs was identified that confer conjugative properties to mycoplasmas, a highly adaptive and diverse group of wall-less bacteria with reduced genomes. Unlike classical ICEs, these mobile elements have no preferential insertion specificity, and multiple mycoplasma ICE copies can be found randomly integrated into the host chromosome. Here, the prototype ICE ofMycoplasma agalactiaewas used to define the minimal conjugative machinery and to propose the first model of ICE transfer in mycoplasmas. This model unveils the complex interactions taking place among coresident ICEs and suggests a way for these elements to extend their influence outside their chromosomal environment. These data pave the way for future studies aiming at deciphering chromosomal transfer, an unconventional mechanism of DNA swapping that has been recently associated with mycoplasma ICEs.

scholarly journals A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
pp. e01226-20
Author(s):  
Hanna Alalam ◽  
Fabrice E. Graf ◽  
Martin Palm ◽  
Marie Abadikhah ◽  
Martin Zackrisson ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Horizontal Transmission ◽  
Antibiotic Resistance Genes ◽  
Host Cells ◽  
The Novel ◽  
Content Type ◽  
Rapid Construction ◽  
Cost Efficient ◽  
Efficient Screening ◽  
New Framework

ABSTRACTThe rapid horizontal transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. Plasmid-carrying donor strains are constructed in high-throughput. We then mix the resistance plasmid-carrying donors with recipients in a design where only transconjugants can reproduce, measure growth in dense intervals, and extract transmission times as the growth lag. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within Escherichia coli populations, by screening the Keio deletion collection in high replication. We recover all seven known chromosomal gene mutants affecting conjugation as donors and identify many novel mutants, all of which diminish antibiotic resistance transmission. We validate nine of the novel genes’ effects in liquid mating assays and complement one of the novel genes’ effect on conjugation (rseA). The new framework holds great potential for exhaustive disclosing of candidate targets for helper drugs that delay resistance development in patients and societies and improve the longevity of current and future antibiotics. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains.IMPORTANCE The rapid transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within E. coli populations. We recover all previously known and many novel chromosomal gene mutants that affect conjugation efficiency. The new framework holds great potential for rapid screening of compounds that decrease transmission. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains.

scholarly journals Use of Proteins Identified through a Functional Genomic Screen To Develop a Protein Subunit Vaccine That Provides Significant Protection against Virulent Streptococcus suis in Pigs

2017 ◽  
Vol 86 (3) ◽  
Author(s):  
Susan L. Brockmeier ◽  
Crystal L. Loving ◽  
Tracy L. Nicholson ◽  
Jinhong Wang ◽  
Sarah E. Peters ◽  
...  
Keyword(s):  
Immune Response ◽  
Vaccine Trial ◽  
Streptococcus Suis ◽  
Protein Subunit ◽  
Content Type ◽  
Degree Of Protection ◽  
Wide Range ◽  
Associated Proteins ◽  
Clinical Protection ◽  
Cellular Immune

ABSTRACT Streptococcus suis is a bacterium that is commonly carried in the respiratory tract and that is also one of the most important invasive pathogens of swine, commonly causing meningitis, arthritis, and septicemia. Due to the existence of many serotypes and a wide range of immune evasion capabilities, efficacious vaccines are not readily available. The selection of S. suis protein candidates for inclusion in a vaccine was accomplished by identifying fitness genes through a functional genomics screen and selecting conserved predicted surface-associated proteins. Five candidate proteins were selected for evaluation in a vaccine trial and administered both intranasally and intramuscularly with one of two different adjuvant formulations. Clinical protection was evaluated by subsequent intranasal challenge with virulent S. suis . While subunit vaccination with the S. suis proteins induced IgG antibodies to each individual protein and a cellular immune response to the pool of proteins and provided substantial protection from challenge with virulent S. suis , the immune response elicited and the degree of protection were dependent on the parenteral adjuvant given. Subunit vaccination induced IgG reactive against different S. suis serotypes, indicating a potential for cross protection.

scholarly journals A novel putative microtubule-associated protein is involved in arbuscule development during arbuscular mycorrhiza formation

2021 ◽  
Author(s):  
Tania Ho-Plágaro ◽  
Raúl Huertas ◽  
María I Tamayo-Navarrete ◽  
Elison Blancaflor ◽  
Nuria Gavara ◽  
...  
Keyword(s):  
Plant Root ◽  
Arbuscular Mycorrhizal ◽  
Host Cells ◽  
Root Cells ◽  
Filament Dynamics ◽  
Fungal Structure ◽  
Genes Encoding ◽  
Associated Proteins ◽  
Mycorrhizal Development

Abstract The formation of arbuscular mycorrhizal (AM) symbiosis requires plant root host cells to undergo major structural and functional reprogramming in order to house the highly branched AM fungal structure for the reciprocal exchange of nutrients. These morphological modifications are associated with cytoskeleton remodelling. However, molecular bases and the role of microtubules (MTs) and actin filament dynamics during AM formation are largely unknown. In this study, the tomato tsb gene, belonging to a Solanaceae group of genes encoding MT-associated proteins for pollen development, was found to be highly expressed in root cells containing arbuscules. At earlier stages of mycorrhizal development, tsb overexpression enhanced the formation of highly developed and transcriptionally active arbuscules, while tsb silencing hampers the formation of mature arbuscules and represses arbuscule functionality. However, at later stages of mycorrhizal colonization, tsb OE roots accumulate fully developed transcriptionally inactive arbuscules, suggesting that the collapse and turnover of arbuscules might be impaired by TSB accumulation. Imaging analysis of the MT cytoskeleton in cortex root cells overexpressing tsb revealed that TSB is involved in MT-bundling. Taken together, our results provide unprecedented insights into the role of novel MT-associated protein in MT rearrangements throughout the different stages of the arbuscule life cycle.

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