scholarly journals Borrelia burgdorferi Needs Chemotaxis To Establish Infection in Mammals and To Accomplish Its Enzootic Cycle

2012 ◽  
Vol 80 (7) ◽  
pp. 2485-2492 ◽  
Author(s):  
Ching Wooen Sze ◽  
Kai Zhang ◽  
Toru Kariu ◽  
Utpal Pal ◽  
Chunhao Li
Keyword(s):  
Borrelia Burgdorferi ◽  
Capillary Tube ◽  
Virulent Strain ◽  
Gene Encoding ◽  
Content Type ◽  
Immunodeficient Mice ◽  
Passage Number ◽  
Low Passage ◽  
Enzootic Cycle

ABSTRACTBorrelia burgdorferi, the causative agent of Lyme disease, can be recovered from different organs of infected animals and patients, indicating that the spirochete is very invasive. Motility and chemotaxis contribute to the invasiveness ofB. burgdorferiand play important roles in the process of the disease. Recent reports have shown that motility is required for establishing infection in mammals. However, the role of chemotaxis in virulence remains elusive. Our previous studies showed thatcheA2, a gene encoding a histidine kinase, is essential for the chemotaxis ofB. burgdorferi. In this report, thecheA2gene was inactivated in a low-passage-number virulent strain ofB. burgdorferi. In vitroanalyses (microscopic observations, computer-based bacterial tracking analysis, swarm plate assays, and capillary tube assays) showed that thecheA2mutant failed to reverse and constantly ran in one direction; the mutant was nonchemotactic to attractants. Mouse needle infection studies showed that thecheA2mutant failed to infect either immunocompetent or immunodeficient mice and was quickly eliminated from the initial inoculation sites. Tick-mouse infection studies revealed that although the mutant was able to survive in ticks, it failed to establish a new infection in mice via tick bites. The altered phenotypes were completely restored when the mutant was complemented. Collectively, these data demonstrate thatB. burgdorferineeds chemotaxis to establish mammalian infection and to accomplish its natural enzootic cycle.

scholarly journals Altered Murine Tissue Colonization by Borrelia burgdorferi following Targeted Deletion of Linear Plasmid 17-Carried Genes

2012 ◽  
Vol 80 (5) ◽  
pp. 1773-1782 ◽  
Author(s):  
Timothy Casselli ◽  
Yvonne Tourand ◽  
Troy Bankhead
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Mutant Strain ◽  
Genetic Manipulation ◽  
Infectious Clone ◽  
Linear Plasmid ◽  
Peripheral Tissues ◽  
Content Type ◽  
Immunodeficient Mice

ABSTRACTThe causative agent of Lyme disease,Borrelia burgdorferi, possesses a segmented genome comprised of a single linear chromosome and upwards of 23 linear and circular plasmids. Much of what is known about plasmid-borne genes comes from studying laboratory clones that have spontaneously lost one or more plasmids duringin vitropassage. Some plasmids, including the linear plasmid lp17, are never or rarely reported to be lost during routine culture; therefore, little is known about the requirement of these conserved plasmids for infectivity. In this study, the effects of deleting regions of lp17 were examined bothin vitroandin vivo. A mutant strain lacking the genesbbd16tobbd25showed no deficiency in the ability to establish infection or disseminate to the bloodstream of mice; however, colonization of peripheral tissues was delayed. Despite the ability to colonize ear, heart, and joint tissues, this mutant exhibited a defect in bladder tissue colonization for up to 56 days postinfection. This phenotype was not observed in immunodeficient mice, suggesting that bladder colonization by the mutant strain was inhibited by an adaptive immune-based mechanism. Moreover, the mutant displayed increased expression of outer surface protein Cin vitro, which was correlated with the absence of the genebbd18. To our knowledge, this is the first report involving genetic manipulation of lp17 in an infectious clone ofB. burgdorferiand reveals for the first time the effects of lp17 gene deletion during murine infection by the Lyme disease spirochete.

scholarly journals Lactic Acid Supplementation Increases Quantity and Quality of Gametocytes in Plasmodium falciparum Culture

2020 ◽  
Vol 89 (1) ◽  
pp. e00635-20
Author(s):  
Rachel West ◽  
David J. Sullivan
Keyword(s):  
Plasmodium Falciparum ◽  
Lactic Acid ◽  
Blood Film ◽  
Content Type ◽  
Acid Supplementation ◽  
Membrane Feeding ◽  
Medium Exchange ◽  
Passage Number ◽  
Low Passage

ABSTRACTMalaria infection by Plasmodium falciparum continues to afflict millions of people worldwide, with transmission being dependent upon mosquito ingestion of the parasite gametocyte stage. These sexually committed stages develop from the asexual stages, yet the factors behind this transition are not completely understood. Here, we found that lactic acid increases gametocyte quantity and quality in P. falciparum culture. Low-passage-number NF54 parasites exposed to 8.2 mM lactic acid for various times were monitored using blood film gametocyte counts and RNA analysis throughout 2 weeks of gametocyte development in vitro for a total of 5 biological cohorts. We found that daily continuous medium exchange and 8.2 mM lactic acid supplementation increased gametocytemia approximately 2- to 6-fold relative to controls after 5 days. In membrane feeding mosquito infection experiments, we found that gametocytes continuously exposed to 8.2 mM lactic acid supplementations were more infectious to Anopheles stephensi mosquitoes, essentially doubling prevalence of infected midguts and oocyst density. Supplementation on days 9 to 16 did not increase the quantity of gametocytes but did increase quality, as measured by oocyst density, by 2.4-fold. Lactic acid did not impact asexual growth, as measured by blood film counts and luciferase quantification, as well as radioactive hypoxanthine incorporation assays. These data indicate a novel role for lactic acid in sexual development of the parasite.

scholarly journals Outer Membrane Proteins BB0405 and BB0406 Are Immunogenic, but Only BB0405 Is Required for Borrelia burgdorferi Infection

2016 ◽  
Vol 85 (2) ◽  
Author(s):  
Binu Shrestha ◽  
Melisha R. Kenedy ◽  
Darrin R. Akins
Keyword(s):  
Borrelia Burgdorferi ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Human Infection ◽  
Factor H ◽  
Serum Resistance ◽  
Gene Encoding ◽  
Content Type ◽  
Protein Factor

ABSTRACT We recently identified the Borrelia burgdorferi outer membrane protein (OMP) BB0406 and found that the gene encoding this OMP was cotranscribed with the gene encoding the OMP BB0405. Interestingly, BB0405 and BB0406 share 59% similarity and are grouped into the same B. burgdorferi paralogous gene family. Given their overall similarity, it is plausible that both OMPs have similar or overlapping functions in this pathogenic spirochete. BB0405 was recently shown to be required for mammalian infection despite the observations that BB0405 is poorly immunogenic and not recognized during mouse or human infection. BB0405 orthologs have also been shown to bind the complement regulator protein factor H. Therefore, to better elucidate the role of BB0405 and its paralog BB0406 during infection and in serum resistance, we examined both proteins in animal infection, factor H binding, and serum sensitivity assays. Our combined results suggest that BB0405- and BB0406-specific antibodies are borreliacidal and that both OMPs are immunogenic during nonhuman primate infection. Additionally, while BB0405 was found to be required for establishing mouse infection, BB0406 was not found to be essential for infectivity. In contrast to data from previous reports, however, neither OMP was found to bind human factor H or to be required for enhancing serum resistance of B. burgdorferi in vitro.

scholarly journals Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

2014 ◽  
Vol 81 (3) ◽  
pp. 1038-1046 ◽  
Author(s):  
Irene N. Kasumba ◽  
Aaron Bestor ◽  
Kit Tilly ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Targeted Mutagenesis ◽  
Multiple Cloning Site ◽  
Stable Gene ◽  
Content Type ◽  
Shuttle Vectors ◽  
Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

scholarly journals Novel regulators of the csgD gene encoding the master regulator of biofilm formation in Escherichia coli K-12

Microbiology ◽  
2020 ◽  
Vol 166 (9) ◽  
pp. 880-890 ◽  
Author(s):  
Hiroshi Ogasawara ◽  
Toshiyuki Ishizuka ◽  
Shuhei Hotta ◽  
Michiko Aoki ◽  
Tomohiro Shimada ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Cell Aggregation ◽  
Master Regulator ◽  
Gene Encoding ◽  
Content Type ◽  
Promoter Probe ◽  
K 12 ◽  
Specific Environmental Condition

Under stressful conditions, Escherichia coli forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the csgD promoter, each monitoring a specific environmental condition or factor. In order to identify the whole set of TFs involved in this typical multi-factor promoter, we performed in this study ‘promoter-specific transcription-factor’ (PS-TF) screening in vitro using a set of 198 purified TFs (145 TFs with known functions and 53 hitherto uncharacterized TFs). A total of 48 TFs with strong binding to the csgD promoter probe were identified, including 35 known TFs and 13 uncharacterized TFs, referred to as Y-TFs. As an attempt to search for novel regulators, in this study we first analysed a total of seven Y-TFs, including YbiH, YdcI, YhjC, YiaJ, YiaU, YjgJ and YjiR. After analysis of curli fimbriae formation, LacZ-reporter assay, Northern-blot analysis and biofilm formation assay, we identified at least two novel regulators, repressor YiaJ (renamed PlaR) and activator YhjC (renamed RcdB), of the csgD promoter.

scholarly journals BrucellaPeriplasmic Protein EipB Is a Molecular Determinant of Cell Envelope Integrity and Virulence

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Julien Herrou ◽  
Jonathan W. Willett ◽  
Aretha Fiebig ◽  
Daniel M. Czyż ◽  
Jason X. Cheng ◽  
...  
Keyword(s):  
Cell Envelope ◽  
Mammalian Host ◽  
Periplasmic Protein ◽  
Periplasmic Space ◽  
Gene Encoding ◽  
Tetratricopeptide Repeats ◽  
Content Type ◽  
Family Protein ◽  
Synthetically Lethal

ABSTRACTThe Gram-negative cell envelope is a remarkable structure with core components that include an inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. Multiple molecular systems function to maintain integrity of this essential barrier between the interior of the cell and its surrounding environment. We show that a conserved DUF1849 family protein, EipB, is secreted to the periplasmic space ofBrucellaspecies, a monophyletic group of intracellular pathogens. In the periplasm, EipB folds into an unusual 14-stranded β-spiral structure that resembles the LolA and LolB lipoprotein delivery system, though the overall fold of EipB is distinct from LolA/LolB. Deletion ofeipBresults in defects inBrucellacell envelope integrityin vitroand in maintenance of spleen colonization in a mouse model ofBrucella abortusinfection. Transposon disruption ofttpA, which encodes a periplasmic protein containing tetratricopeptide repeats, is synthetically lethal witheipBdeletion.ttpAis a reported virulence determinant inBrucella, and our studies ofttpAdeletion and overexpression strains provide evidence that this gene also contributes to cell envelope function. We conclude thateipBandttpAfunction in theBrucellaperiplasmic space to maintain cell envelope integrity, which facilitates survival in a mammalian host.IMPORTANCEBrucellaspecies cause brucellosis, a global zoonosis. A gene encoding a conserved DUF1849-family protein, which we have named EipB, is present in all sequencedBrucellaand several other genera in the classAlphaproteobacteria. The manuscript provides the first functional and structural characterization of a DUF1849 protein. We show that EipB is secreted to the periplasm where it forms a spiral-shaped antiparallel β protein that is a determinant of cell envelope integrityin vitroand virulence in an animal model of disease.eipBgenetically interacts withttpA, which also encodes a periplasmic protein. We propose that EipB and TtpA function as part of a system required for cell envelope homeostasis in selectAlphaproteobacteria.

scholarly journals Role of the GapA and CrmA Cytadhesins of Mycoplasma gallisepticum in Promoting Virulence and Host Colonization

2013 ◽  
Vol 81 (5) ◽  
pp. 1618-1624 ◽  
Author(s):  
Ivana Indiková ◽  
Peter Much ◽  
László Stipkovits ◽  
Karin Siebert-Gulle ◽  
Michael P. Szostak ◽  
...  
Keyword(s):  
Point Mutation ◽  
Mycoplasma Gallisepticum ◽  
Chronic Respiratory Disease ◽  
Host Colonization ◽  
Content Type ◽  
Inner Organs ◽  
Avian Pathogen ◽  
Low Passage

ABSTRACTMycoplasma gallisepticumis an important avian pathogen that commonly induces chronic respiratory disease in chicken. To better understand the mycoplasma factors involved in host colonization, chickens were infected via aerosol with two hemadsorption-negative (HA−) mutants, mHAD3 and RCL2, that were derived from a low passage of the pathogenic strain R (Rlow) and are both deficient in the two major cytadhesins GapA and CrmA. After 9 days of infection, chickens were monitored for air sac lesions and for the presence of mycoplasmas in various organs. The data showed that mHAD3, in which thecrmAgene has been disrupted, did not promote efficient colonization or significant air sac lesions. In contrast, the spontaneous HA−RCL2 mutant, which contains a point mutation in thegapAstructural gene, successfully colonized the respiratory tract and displayed an attenuated virulence compared to that of Rlow. It has previously been shownin vitrothat the point mutation of RCL2 spontaneously reverts with a high frequency, resulting in on-and-off switching of the HA phenotype. Detailed analyses further revealed that such an event is not responsible for the observedin vivooutcome, since 98.4% of the mycoplasma populations recovered from RCL2-infected chickens still display the mutation and the associated phenotype. Unlike Rlow, however, RCL2 was unable to colonize inner organs. These findings demonstrate the major role played by the GapA and CrmA proteins inM. gallisepticumhost colonization and virulence.

scholarly journals Expression of Adhesive Pili and the Collagen-Binding Adhesin Ace Is Activated by ArgR Family Transcription Factors inEnterococcus faecalis

2018 ◽  
Vol 200 (18) ◽  
Author(s):  
Dawn A. Manias ◽  
Gary M. Dunny
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Enterococcus Faecalis ◽  
Opportunistic Infections ◽  
Structural Genes ◽  
Gene Encoding ◽  
Collagen Binding ◽  
Content Type ◽  
Surface Adhesins

ABSTRACTIt was shown previously that the disruption of theahrCgene encoding a predicted ArgR family transcription factor results in a severe defect in biofilm formationin vitro, as well as a significant attenuation of virulence ofEnterococcus faecalisstrain OG1RF in multiple experimental infection models. Using transcriptome sequencing (RNA-seq), we observedahrC-dependent changes in the expression of more than 20 genes. AhrC-repressed genes included predicted determinants of arginine catabolism and several other metabolic genes and predicted transporters, while AhrC-activated genes included determinants involved in the production of surface protein adhesins. Most notably, the structural and regulatory genes of theebplocus encoding adhesive pili were positively regulated, as well as theacegene, encoding a collagen-binding adhesin. UsinglacZtranscription reporter fusions, we determined thatahrCand a secondargRtranscription factor gene,argR2, both function to activate the expression ofebpR, which directly activates the transcription of the pilus structural genes. Our data suggest that in the wild-typeE. faecalis, the low levels of EbpR limit the expression of pili and that biofilm biomass is also limited by the amount of pili expressed by the bacteria. The expression ofaceis similarly enhanced by AhrC and ArgR2, butaceexpression is not dependent on EbpR. Our results demonstrate the existence of novel regulatory cascades controlled by a pair of ArgR family transcription factors that might function as a heteromeric protein complex.IMPORTANCECell surface adhesins play critical roles in the formation of biofilms, host colonization, and the pathogenesis of opportunistic infections byEnterococcus faecalis. Here, we present new results showing that the expression of two major enterococcal surface adhesins,ebppili, and the collagen-binding protein Ace is positively regulated at the transcription level by twoargRfamily transcription factors, AhrC and ArgR2. In the case of pili, the direct target of regulation is theebpRgene, previously shown to activate the transcription of the pilus structural genes, while the activation ofacetranscription appears to be directly impacted by the two ArgR proteins. These transcription factors may represent new targets for blocking enterococcal infections.

scholarly journals Reduced Immune Response to Borrelia burgdorferi in the Absence of γδ T Cells

2011 ◽  
Vol 79 (10) ◽  
pp. 3940-3946 ◽  
Author(s):  
Cuixia Shi ◽  
Bikash Sahay ◽  
Jennifer Q. Russell ◽  
Karen A. Fortner ◽  
Nicholas Hardin ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Borrelia Burgdorferi ◽  
Adaptive Immune Response ◽  
Γδ T Cells ◽  
Content Type ◽  
Adaptive Immune ◽  
Cytokines And Chemokines

ABSTRACTLittle is known regarding the function of γδ T cells, although they accumulate at sites of inflammation in infections and autoimmune disorders. We previously observed that γδ T cellsin vitroare activated byBorrelia burgdorferiin a TLR2-dependent manner. We now observe that the activated γδ T cells can in turn stimulate dendritic cellsin vitroto produce cytokines and chemokines that are important for the adaptive immune response. This suggested thatin vivoγδ T cells may assist in activating the adaptive immune response. We examined this possibilityin vivoand observed that γδ T cells are activated and expand in number duringBorreliainfection, and this was reduced in the absence of TLR2. Furthermore, in the absence of γδ T cells, there was a significantly blunted response of adaptive immunity, as reflected in reduced expansion of T and B cells and reduced serum levels of anti-Borreliaantibodies, cytokines, and chemokines. This paralleled a greaterBorreliaburden in γδ-deficient mice as well as more cardiac inflammation. These findings are consistent with a model of γδ T cells functioning to promote the adaptive immune response during infection.

scholarly journals Evaluation of RevA, a Fibronectin-Binding Protein of Borrelia burgdorferi, as a Potential Vaccine Candidate for Lyme Disease

2013 ◽  
Vol 20 (6) ◽  
pp. 892-899 ◽  
Author(s):  
Angela M. Floden ◽  
Tammy Gonzalez ◽  
Robert A. Gaultney ◽  
Catherine A. Brissette
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Passive Immunization ◽  
Surface Protein ◽  
Surface Expression ◽  
Content Type ◽  
Good Target ◽  
Potential Vaccine ◽  
Mammalian Infection

ABSTRACTPrevious studies indicated that the Lyme disease spirocheteBorrelia burgdorferiexpresses the RevA outer surface protein during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA appears to be a good target for preventive therapies. RevA proteins are highly conserved across all Lyme borreliae, and antibodies against RevA protein are cross-reactive among RevA proteins from diverse strains. Mice infected withB. burgdorferimounted a rapid IgM response to RevA, followed by a strong IgG response that generally remained elevated for more than 12 months, suggesting continued exposure of RevA protein to the immune system. RevA antibodies were bactericidalin vitro. To evaluate the RevA antigen as a potential vaccine, mice were vaccinated with recombinant RevA and challenged withB. burgdorferiby inoculation with a needle or by a tick bite. Cultured tissues from all treatment groups were positive forB. burgdorferi. Vaccinated animals also appeared to have similar levels ofB. burgdorferiDNA compared to nonvaccinated controls. Despite its antigenicity, surface expression, and the production of bactericidal antibodies against it, RevA does not protect againstBorrelia burgdorferiinfection in a mouse model. However, passive immunization with anti-RevA antibodies did prevent infection, suggesting the possible utility of RevA-based immunotherapeutics or vaccine.

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