scholarly journals Nonselective Persistence of a Rickettsia conorii Extrachromosomal Plasmid during Mammalian Infection

2016 ◽  
Vol 84 (3) ◽  
pp. 790-797 ◽  
Author(s):  
Sean P. Riley ◽  
Abigail I. Fish ◽  
Daniel A. Garza ◽  
Kaikhushroo H. Banajee ◽  
Emma K. Harris ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Genetic Manipulation ◽  
Fluorescent Proteins ◽  
Spotted Fever ◽  
Rickettsia Conorii ◽  
Mediterranean Spotted Fever ◽  
Extrachromosomal Dna ◽  
Content Type

Scientific analysis of the genusRickettsiais undergoing a rapid period of change with the emergence of viable genetic tools. The development of these tools for the mutagenesis of pathogenic bacteria will permit forward genetic analysis ofRickettsiapathogenesis. Despite these advances, uncertainty still remains regarding the use of plasmids to study these bacteria inin vivomammalian models of infection, namely, the potential for virulence changes associated with the presence of extrachromosomal DNA and nonselective persistence of plasmids in mammalian models of infection. Here, we describe the transformation ofRickettsia conoriiMalish 7 with the plasmid pRam18dRGA[AmTrCh]. TransformedR. conoriistably maintains this plasmid in infected cell cultures, expresses the encoded fluorescent proteins, and exhibits growth kinetics in cell culture similar to those of nontransformedR. conorii. Using a well-established murine model of fatal Mediterranean spotted fever, we demonstrate thatR. conorii(pRam18dRGA[AmTrCh]) elicits the same fatal outcomes in animals as its untransformed counterpart and, importantly, maintains the plasmid throughout infection in the absence of selective antibiotic pressure. Interestingly, plasmid-transformedR. conoriiwas readily observed both in endothelial cells and within circulating leukocytes. Together, our data demonstrate that the presence of an extrachromosomal DNA element in a pathogenic rickettsial species does not affect eitherin vitroproliferation orin vivoinfectivity in models of disease and that plasmids such as pRam18dRGA[AmTrCh] are valuable tools for the further genetic manipulation of pathogenic rickettsiae.

scholarly journals Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Desmond A. Moore ◽  
Zakiya N. Whatley ◽  
Chandra P. Joshi ◽  
Masaki Osawa ◽  
Harold P. Erickson
Keyword(s):  
Cell Division ◽  
Fluorescent Proteins ◽  
Sole Source ◽  
Ring Structure ◽  
Content Type ◽  
Z Ring ◽  
Complete Cell ◽  
The Right

ABSTRACT FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo. One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro. Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques. IMPORTANCE One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging.

scholarly journals The HtrA-Like Protease CD3284 Modulates Virulence of Clostridium difficile

2014 ◽  
Vol 82 (10) ◽  
pp. 4222-4232 ◽  
Author(s):  
Dennis Bakker ◽  
Anthony M. Buckley ◽  
Anne de Jong ◽  
Vincent J. C. van Winden ◽  
Joost P. A. Verhoeks ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Virulence Factors ◽  
Pathogenic Bacteria ◽  
Microarray Data Analysis ◽  
Toxin A ◽  
Hamster Model ◽  
Golden Syrian Hamster ◽  
Content Type

ABSTRACTIn the past decade,Clostridium difficilehas emerged as an important gut pathogen. Symptoms ofC. difficileinfection range from mild diarrhea to pseudomembranous colitis. Besides the two main virulence factors toxin A and toxin B, other virulence factors are likely to play a role in the pathogenesis of the disease. In other Gram-positive and Gram-negative pathogenic bacteria, conserved high-temperature requirement A (HtrA)-like proteases have been shown to have a role in protein homeostasis and quality control. This affects the functionality of virulence factors and the resistance of bacteria to (host-induced) environmental stresses. We found that theC. difficile630 genome encodes a single HtrA-like protease (CD3284; HtrA) and have analyzed its rolein vivoandin vitrothrough the creation of an isogenic ClosTron-basedhtrAmutant ofC. difficilestrain 630Δerm(wild type). In contrast to the attenuated phenotype seen withhtrAdeletion in other pathogens, this mutant showed enhanced virulence in the Golden Syrian hamster model of acuteC. difficileinfection. Microarray data analysis showed a pleiotropic effect ofhtrAon the transcriptome ofC. difficile, including upregulation of the toxin A gene. In addition,the htrAmutant showed reduced spore formation and adherence to colonic cells. Together, our data show thathtrAcan modulate virulence inC. difficile.

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

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
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.

scholarly journals Involvement of the AcrAB-TolC Efflux Pump in the Resistance, Fitness, and Virulence of Enterobacter cloacae

2012 ◽  
Vol 56 (4) ◽  
pp. 2084-2090 ◽  
Author(s):  
Astrid Pérez ◽  
Margarita Poza ◽  
Ana Fernández ◽  
Maria del Carmen Fernández ◽  
Susana Mallo ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Pathogenic Bacteria ◽  
Efflux Pump ◽  
Enterobacter Cloacae ◽  
Efflux Pumps ◽  
Clinical Isolates ◽  
Wild Type ◽  
Content Type

ABSTRACTMultidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence inEnterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates ofE. cloacaewere used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). TheacrAandtolCgenes were deleted in strains EcDC64 and Jc194 to produce, respectively, EcΔacrAand EcΔtolCand JcΔacrAand JcΔtolCknockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance ofE. cloacaeto several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that theacrAandtolCgenes both affect the fitness ofE. cloacae, as fitness was clearly reduced in theacrAandtolCKO strains. The median CI values obtainedin vitroandin vivowere, respectively, 0.42 and 0.3 for EcDC64/EcΔacrA, 0.24 and 0.38 for EcDC64/EcΔtolC, 0.15 and 0.11 for Jc194/JcΔacrA, and 0.38 and 0.39 for Jc194/JcΔtolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in bothE. cloacaeclinical strains when either theacrAortolCgene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates ofE. cloacae.

scholarly journals Demonstration of Rickettsia conorii-induced endothelial injury in vivo by measuring circulating endothelial cells, thrombomodulin, and von Willebrand factor in patients with Mediterranean spotted fever [see comments]

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2109-2116
Author(s):  
F George ◽  
P Brouqui ◽  
MC Boffa ◽  
M Mutin ◽  
M Drancourt ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Spotted Fever ◽  
Clinical Manifestations ◽  
Endothelial Injury ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rickettsia Conorii ◽  
Mediterranean Spotted Fever ◽  
Von Willebrand ◽  
Willebrand Factor

The endothelial cell (EC) is the primary target for Rickettsia conorii (RC) in Mediterranean spotted fever (MSF). Clinical manifestations such as thrombosis and vasculitis are mediated by pathologic changes localized in blood vessels. To study the in vivo endothelial injury induced by RC, markers of endothelial damage, including circulating EC (CEC), plasmatic thrombomodulin (TM), and von Willebrand factor (vWF), were investigated in 12 patients with MSF. CEC were counted in whole blood by a new immunomagnetic separation assay using a specific anti-EC antibody, S-Endo 1. Plasmatic TM and vWF antigens were measured by enzyme-linked immunosorbent assay. High levels of CEC and cell fragments were found in patients with a severe or malignant form of MSF. Sequential studies of CEC showed a decrease from 162 +/- 454 cells/mL before treatment to 6 +/- 7 cells/mL during treatment and recovery. Mean plasma TM and vWF levels that were also elevated before therapy (TM, 106 +/- 27 ng/mL; vWF, 420% +/- 164%) decreased progressively (TM, 55 +/- 43 ng/mL; vWF, 148% +/- 26%) during treatment. The measurement of cellular and molecular markers of vascular damage such as CEC, plasmatic TM, and vWF contributes to the definition of the Rickettsia-induced endothelial injury in vivo.

scholarly journals Evolutionary Adaptation of an AraC-Like Regulatory Protein in Citrobacter rodentium and Escherichia Species

2015 ◽  
Vol 83 (4) ◽  
pp. 1384-1395 ◽  
Author(s):  
Aimee Tan ◽  
Nicola K. Petty ◽  
Dianna Hocking ◽  
Vicki Bennett-Wood ◽  
Matthew Wakefield ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Regulatory Protein ◽  
Gene Repertoire ◽  
Citrobacter Rodentium ◽  
Environmental Strain ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding

The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogenCitrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and thegrlRAoperon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genusEscherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated thatregAhas been horizontally transferred toEscherichiaspp. andC. rodentium. Comparative studies of two RegA homologues, namely, those fromC. rodentiumandE. coliSMS-3-5, a multiresistant environmental strain ofE. coli, showed that the two regulators acted similarlyin vitrobut differed in terms of their abilities to activate the virulence ofC. rodentiumin vivo, which evidently was due to their differential activation ofgrlRA. Our data indicate that RegA fromC. rodentiumhas strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence byC. rodentiumand on the evolution of virulence-regulatory genes of bacterial pathogens in general.

scholarly journals Demonstration of Rickettsia conorii-induced endothelial injury in vivo by measuring circulating endothelial cells, thrombomodulin, and von Willebrand factor in patients with Mediterranean spotted fever [see comments]

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2109-2116 ◽  
Author(s):  
F George ◽  
P Brouqui ◽  
MC Boffa ◽  
M Mutin ◽  
M Drancourt ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Spotted Fever ◽  
Clinical Manifestations ◽  
Endothelial Injury ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rickettsia Conorii ◽  
Mediterranean Spotted Fever ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The endothelial cell (EC) is the primary target for Rickettsia conorii (RC) in Mediterranean spotted fever (MSF). Clinical manifestations such as thrombosis and vasculitis are mediated by pathologic changes localized in blood vessels. To study the in vivo endothelial injury induced by RC, markers of endothelial damage, including circulating EC (CEC), plasmatic thrombomodulin (TM), and von Willebrand factor (vWF), were investigated in 12 patients with MSF. CEC were counted in whole blood by a new immunomagnetic separation assay using a specific anti-EC antibody, S-Endo 1. Plasmatic TM and vWF antigens were measured by enzyme-linked immunosorbent assay. High levels of CEC and cell fragments were found in patients with a severe or malignant form of MSF. Sequential studies of CEC showed a decrease from 162 +/- 454 cells/mL before treatment to 6 +/- 7 cells/mL during treatment and recovery. Mean plasma TM and vWF levels that were also elevated before therapy (TM, 106 +/- 27 ng/mL; vWF, 420% +/- 164%) decreased progressively (TM, 55 +/- 43 ng/mL; vWF, 148% +/- 26%) during treatment. The measurement of cellular and molecular markers of vascular damage such as CEC, plasmatic TM, and vWF contributes to the definition of the Rickettsia-induced endothelial injury in vivo.

scholarly journals A Two-Step Mechanism for the Activation of Actinorhodin Export and Resistance in Streptomyces coelicolor

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ye Xu ◽  
Andrew Willems ◽  
Catherine Au-yeung ◽  
Kapil Tahlan ◽  
Justin R. Nodwell
Keyword(s):  
Secondary Metabolites ◽  
Pathogenic Bacteria ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Resistance Mechanisms ◽  
Biosynthetic Gene ◽  
Content Type

ABSTRACT Many microorganisms produce secondary metabolites that have antibiotic activity. To avoid self-inhibition, the producing cells often encode cognate export and/or resistance mechanisms in the biosynthetic gene clusters for these molecules. Actinorhodin is a blue-pigmented antibiotic produced by Streptomyces coelicolor. The actAB operon, carried in the actinorhodin biosynthetic gene cluster, encodes two putative export pumps and is regulated by the transcriptional repressor protein ActR. In this work, we show that normal actinorhodin yields require actAB expression. Consistent with previous in vitro work, we show that both actinorhodin and its 3-ring biosynthetic intermediates [e.g., (S)-DNPA] can relieve repression of actAB by ActR in vivo. Importantly, an ActR mutant that interacts productively with (S)-DNPA but not with actinorhodin responds to the actinorhodin biosynthetic pathway with the induction of actAB and normal yields of actinorhodin. This suggests that the intermediates are sufficient to trigger the export genes in actinorhodin-producing cells. We further show that actinorhodin-producing cells can induce actAB expression in nonproducing cells; however, in this case actinorhodin is the most important signal. Finally, while the “intermediate-only” ActR mutant permits sufficient actAB expression for normal actinorhodin yields, this expression is short-lived. Sustained culture-wide expression requires a subsequent actinorhodin-mediated signaling step, and the defect in this response causes widespread cell death. These results are consistent with a two-step model for actinorhodin export and resistance where intermediates trigger initial expression for export from producing cells and actinorhodin then triggers sustained export gene expression that confers culture-wide resistance. IMPORTANCE Understanding the links between antibiotic resistance and biosynthesis is important for our efforts to manipulate secondary metabolism. For example, many secondary metabolites are produced at low levels; our work suggests that manipulating export might be one way to enhance yields of these molecules. It also suggests that understanding resistance will be relevant to the generation of novel secondary metabolites through the creation of synthetic secondary metabolic gene clusters. Finally, these cognate resistance mechanisms are related to mechanisms that arise in pathogenic bacteria, and understanding them is relevant to our ability to control microbial infections clinically.

scholarly journals The Borrelia hermsii Factor H Binding Protein FhbA Is Not Required for Infectivity in Mice or for Resistance to Human ComplementIn Vitro

2014 ◽  
Vol 82 (8) ◽  
pp. 3324-3332 ◽  
Author(s):  
Lindy M. Fine ◽  
Daniel P. Miller ◽  
Katherine L. Mallory ◽  
Brittney K. Tegels ◽  
Christopher G. Earnhart ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Binding Protein ◽  
Factor H ◽  
Negative Regulator ◽  
Human Complement ◽  
Relapsing Fever ◽  
Borrelia Hermsii ◽  
Content Type

ABSTRACTThe primary causative agent of tick-borne relapsing fever in North America isBorrelia hermsii. It has been hypothesized thatB. hermsiievades complement-mediated destruction by binding factor H (FH), a host-derived negative regulator of complement.In vitro,B. hermsiiproduces a single FH binding protein designated FhbA (FH binding protein A). The properties and ligand binding activity of FhbA suggest that it plays multiple roles in pathogenesis. It binds plasminogen and has been identified as a significant target of a B1b B cell-mediated IgM response in mice. FhbA has also been explored as a potential diagnostic antigen forB. hermsiiinfection in humans. The ability to test the hypothesis that FhbA is a critical virulence factorin vivohas been hampered by the lack of well-developed systems for the genetic manipulation of the relapsing fever spirochetes. In this report, we have successfully generated aB. hermsiifhbAdeletion mutant (theB. hermsiiYORΔfhbAstrain) through allelic exchange mutagenesis. Deletion offhbAabolished FH binding by the YORΔfhbAstrain and eliminated cleavage of C3b on the cell surface. However, the YORΔfhbAstrain remained infectious in mice and retained resistance to killingin vitroby human complement. Collectively, these results indicate thatB. hermsiiemploys an FhbA/FH-independent mechanism of complement evasion that allows for resistance to killing by human complement and persistence in mice.

scholarly journals Clearance of Pseudomonas aeruginosa Foreign-Body Biofilm Infections through Reduction of the Cyclic Di-GMP Level in the Bacteria

2013 ◽  
Vol 81 (8) ◽  
pp. 2705-2713 ◽  
Author(s):  
Louise D. Christensen ◽  
Maria van Gennip ◽  
Morten T. Rybtke ◽  
Hong Wu ◽  
Wen-Chi Chiang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Silicone Implants ◽  
Content Type ◽  
Biofilm Control ◽  
Secondary Messenger ◽  
Diguanosine Monophosphate ◽  
Opportunistic Pathogenic

ABSTRACTOpportunistic pathogenic bacteria can engage in biofilm-based infections that evade immune responses and develop into chronic conditions. Because conventional antimicrobials cannot efficiently eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. It has recently been established that the secondary messenger cyclic diguanosine monophosphate (c-di-GMP) functions as a positive regulator of biofilm formation in several different bacteria. In the present study we investigated whether manipulation of the c-di-GMP level in bacteria potentially can be used for biofilm controlin vivo. We constructed aPseudomonas aeruginosastrain in which a reduction in the c-di-GMP level can be achieved via induction of theEscherichia coliYhjH c-di-GMP phosphodiesterase. Initial experiments showed that induction ofyhjHexpression led to dispersal of the majority of the bacteria inin vitro-grownP. aeruginosabiofilms. Subsequently, we demonstrated thatP. aeruginosabiofilms growing on silicone implants, located in the peritoneal cavity of mice, dispersed after induction of the YhjH protein. Bacteria accumulated temporarily in the spleen after induction of biofilm dispersal, but the mice tolerated the dispersed bacteria well. The present work provides proof of the concept that modulation of the c-di-GMP level in bacteria is a viable strategy for biofilm control.

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