scholarly journals Selective Association of Outer Surface Lipoproteins with the Lipid Rafts of Borrelia burgdorferi

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Alvaro Toledo ◽  
Jameson T. Crowley ◽  
James L. Coleman ◽  
Timothy J. LaRocca ◽  
Salvatore Chiantia ◽  
...  
Keyword(s):  
Life Cycle ◽  
Borrelia Burgdorferi ◽  
Lipid Rafts ◽  
Outer Surface ◽  
Single Gene ◽  
Functional Adaptation ◽  
Deletion Mutants ◽  
Wild Type ◽  
Content Type ◽  
Single Deletion

ABSTRACTBorrelia burgdorfericontains unique cholesterol-glycolipid-rich lipid rafts that are associated with lipoproteins. These complexes suggest the existence of macromolecular structures that have not been reported for prokaryotes. Outer surface lipoproteins OspA, OspB, and OspC were studied for their participation in the formation of lipid rafts. Single-gene deletion mutants with deletions of ∆ospA, ∆ospB, and ∆ospCand a spontaneous gene mutant, strain B313, which does not express OspA and OspB, were used to establish their structural roles in the lipid rafts. All mutant strains used in this study produced detergent-resistant membranes, a common characteristic of lipid rafts, and had similar lipid and protein slot blot profiles. Lipoproteins OspA and OspB but not OspC were shown to be associated with lipid rafts by transmission electron microscopy. When the ability to form lipid rafts in liveB. burgdorferispirochetes was measured byfluorescenceresonanceenergytransfer (FRET), strain B313 showed a statistically significant lower level of segregation into ordered and disordered membrane domains than did the wild-type and the other single-deletion mutants. The transformation of a B313 strain with a shuttle plasmid containingospArestored the phenotype shared by the wild type and the single-deletion mutants, demonstrating that OspA and OspB have redundant functions. In contrast, a transformed B313 overexpressing OspC neither rescued the FRET nor colocalized with the lipid rafts. Because these lipoproteins are expressed at different stages of the life cycle ofB. burgdorferi, their selective association is likely to have an important role in the structure of prokaryotic lipid rafts and in the organism’s adaptation to changing environments.IMPORTANCELipid rafts are cholesterol-rich clusters within the membranes of cells. Lipid rafts contain proteins that have functions in sensing the cell environment and transmitting signals. Although selective proteins are present in lipid rafts, little is known about their structural contribution to these domains.Borrelia burgdorferi, the agent of Lyme disease, has lipid rafts, which are novel structures in bacteria. Here, we have shown that the raft-associated lipoproteins OspA and OspB selectively contribute to lipid rafts. A similar but non-raft-associated lipoprotein, OspC, cannot substitute for the role of OspA and OspB. In this study, we have demonstrated that lipoprotein association with lipid rafts is selective, further suggesting a functional adaptation to different stages of the spirochete life cycle. The results of this study are of broader importance and can serve as a model for other bacteria that also possess cholesterol in their membranes and, therefore, may share lipid raft traits withBorrelia.

scholarly journals Glutathione Synthesis Contributes to Virulence of Streptococcus agalactiae in a Murine Model of Sepsis

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Elizabeth A. Walker ◽  
Gary C. Port ◽  
Michael G. Caparon ◽  
Blythe E. Janowiak
Keyword(s):  
Streptococcus Agalactiae ◽  
De Novo ◽  
Single Gene ◽  
Neonatal Meningitis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Glutathione Synthesis ◽  
Content Type ◽  
Resistance Pathway

ABSTRACT Streptococcus agalactiae, a leading cause of sepsis and meningitis in neonates, utilizes multiple virulence factors to survive and thrive within the human host during an infection. Unique among the pathogenic streptococci, S. agalactiae uses a bifunctional enzyme encoded by a single gene (gshAB) to synthesize glutathione (GSH), a major antioxidant in most aerobic organisms. Since S. agalactiae can also import GSH, similar to all other pathogenic streptococcal species, the contribution of GSH synthesis to the pathogenesis of S. agalactiae disease is not known. In the present study, gshAB deletion mutants were generated in strains representing three of the most prevalent clinical serotypes of S. agalactiae and were compared against isogenic wild-type and gshAB knock-in strains. When cultured in vitro in a chemically defined medium under nonstress conditions, each mutant and its corresponding wild type had comparable growth rates, generation times, and growth yields. However, gshAB deletion mutants were found to be more sensitive than wild-type or gshAB knock-in strains to killing and growth inhibition by several different reactive oxygen species. Furthermore, deletion of gshAB in S. agalactiae strain COH1 significantly attenuated virulence compared to the wild-type or gshAB knock-in strains in a mouse model of sepsis. Taken together, these data establish that GSH is a virulence factor important for resistance to oxidative stress and that de novo GSH synthesis plays a crucial role in S. agalactiae pathogenesis and further suggest that the inhibition of GSH synthesis may provide an opportunity for the development of novel therapies targeting S. agalactiae disease. IMPORTANCE Approximately 10 to 30% of women are naturally and asymptomatically colonized by Streptococcus agalactiae. However, transmission of S. agalactiae from mother to newborn during vaginal birth is a leading cause of neonatal meningitis. Although colonized mothers who are at risk for transmission to the newborn are treated with antibiotics prior to delivery, S. agalactiae is becoming increasingly resistant to current antibiotic therapies, and new treatments are needed. This research reveals a critical stress resistance pathway, glutathione synthesis, that is utilized by S. agalactiae and contributes to its pathogenesis. Understanding the role of this unique bifunctional glutathione synthesis enzyme in S. agalactiae during sepsis may help elucidate why S. agalactiae produces such an abundance of glutathione compared to other bacteria.

scholarly journals Utilization of Host Iron Sources by Corynebacterium diphtheriae: Multiple Hemoglobin-Binding Proteins Are Essential for the Use of Iron from the Hemoglobin-Haptoglobin Complex

2014 ◽  
Vol 197 (3) ◽  
pp. 553-562 ◽  
Author(s):  
Courtni E. Allen ◽  
Michael P. Schmitt
Keyword(s):  
Double Mutant ◽  
Biological Function ◽  
Corynebacterium Diphtheriae ◽  
Enzyme Linked Immunosorbent Assay ◽  
Deletion Mutants ◽  
Wild Type ◽  
Content Type ◽  
Iron Source ◽  
Hemoglobin Binding ◽  
Single Deletion

The use of hemin iron byCorynebacterium diphtheriaerequires the DtxR- and iron-regulated ABC hemin transporter HmuTUV and the secreted Hb-binding protein HtaA. We recently described two surface anchored proteins, ChtA and ChtC, which also bind hemin and Hb. ChtA and ChtC share structural similarities to HtaA; however, a function for ChtA and ChtC was not determined. In this study, we identified additional host iron sources that are utilized byC. diphtheriae. We show that severalC. diphtheriaestrains use the hemoglobin-haptoglobin (Hb-Hp) complex as an iron source. We report that anhtaAdeletion mutant ofC. diphtheriaestrain 1737 is unable to use the Hb-Hp complex as an iron source, and we further demonstrate that achtA-chtCdouble mutant is also unable to use Hb-Hp iron. Single-deletion mutants ofchtAorchtCuse Hb-Hp iron in a manner similar to that of the wild type. These findings suggest that both HtaA and either ChtA or ChtC are essential for the use of Hb-Hp iron. Enzyme-linked immunosorbent assay (ELISA) studies show that HtaA binds the Hb-Hp complex, and the substitution of a conserved tyrosine (Y361) for alanine in HtaA results in significantly reduced binding.C. diphtheriaewas also able to use human serum albumin (HSA) and myoglobin (Mb) but not hemopexin as iron sources. These studies identify a biological function for the ChtA and ChtC proteins and demonstrate that the use of the Hb-Hp complex as an iron source byC. diphtheriaerequires multiple iron-regulated surface components.

scholarly journals Analysis of the Borrelia burgdorferi Cyclic-di-GMP-Binding Protein PlzA Reveals a Role in Motility and Virulence

2011 ◽  
Vol 79 (5) ◽  
pp. 1815-1825 ◽  
Author(s):  
Joshua E. Pitzer ◽  
Syed Z. Sultan ◽  
Yoshihiro Hayakawa ◽  
Gerry Hobbs ◽  
Michael R. Miller ◽  
...  
Keyword(s):  
Life Cycle ◽  
Borrelia Burgdorferi ◽  
Binding Protein ◽  
Survival Rates ◽  
Wild Type ◽  
Content Type ◽  
Infectious Dose ◽  
Swimming Pattern ◽  
Bacteria Inactivation ◽  
Mutant Cells

ABSTRACTThe cyclic-dimeric-GMP (c-di-GMP)-binding protein PilZ has been implicated in bacterial motility and pathogenesis. Although BB0733 (PlzA), the only PilZ domain-containing protein inBorrelia burgdorferi, was reported to bind c-di-GMP, neither its role in motility or virulence nor it's affinity for c-di-GMP has been reported. We determined that PlzA specifically binds c-di-GMP with high affinity (dissociation constant [Kd], 1.25 μM), consistent withKdvalues reported for c-di-GMP-binding proteins from other bacteria. Inactivation of the monocistronically transcribedplzAresulted in an opaque/solid colony morphology, whereas the wild-type colonies were translucent. While the swimming pattern of mutant cells appeared normal, on swarm plates, mutant cells exhibited a significantly reduced swarm diameter, demonstrating a role ofplzAin motility. Furthermore, theplzAmutant cells were significantly less infectious in experimental mice (as determined by 50% infectious dose [ID50]) relative to wild-type spirochetes. The mutant also had survival rates in fed ticks lower than those of the wild type. Consequently,plzAmutant cells failed to complete the mouse-tick-mouse infection cycle, indicatingplzAis essential for the enzootic life cycle ofB. burgdorferi. All of these defects were corrected when the mutant was complemented incis. We propose that failure ofplzAmutant cells to infect mice was due to altered motility; however, the possibility that an unidentified factor(s) contributed to interruption of theB. burgdorferienzootic life cycle cannot yet be excluded.

scholarly journals Borrelia burgdorferi CheD Promotes Various Functions in Chemotaxis and the Pathogenic Life Cycle of the Spirochete

2016 ◽  
Vol 84 (6) ◽  
pp. 1743-1752 ◽  
Author(s):  
Ki Hwan Moon ◽  
Gerry Hobbs ◽  
M. A. Motaleb
Keyword(s):  
Life Cycle ◽  
Borrelia Burgdorferi ◽  
Phosphatase Activity ◽  
Response Regulator ◽  
Wild Type ◽  
Content Type ◽  
Mouse Infection ◽  
Protein Receptors ◽  
Chemotaxis Proteins

Borrelia burgdorferipossesses a sophisticated chemotaxis signaling system; however, the roles of the majority of the chemotaxis proteins in the infectious life cycle have not yet been demonstrated. Specifically, the role of CheD during host colonization has not been demonstrated in any bacterium. Here, we systematically characterized theB. burgdorferiCheD homolog using genetics and biochemical and mouse-tick-mouse infection cycle studies.Bacillus subtilisCheD plays an important role in chemotaxis by deamidation of methyl-accepting chemotaxis protein receptors (MCPs) and by increasing the receptor kinase activity or enhancing CheC phosphatase activity, thereby regulating the levels of the CheY response regulator. Our biochemical analysis indicates thatB. burgdorferiCheD significantly enhances CheX phosphatase activity by specifically interacting with the phosphatase. Moreover, CheD specifically binds two of the six MCPs, indicating that CheD may also modulate the receptor proteins. Although the motility of thecheDmutant cells was indistinguishable from that of the wild-type cells, the mutant did exhibit reduced chemotaxis. Importantly, the mutant showed significantly reduced infectivity in C3H/HeN mice via needle inoculation. Mouse-tick-mouse infection assays indicated that CheD is dispensable for acquisition or transmission of spirochetes; however, the viability ofcheDmutants in ticks is marginally reduced compared to that of the wild-type or complementedcheDspirochetes. These data suggest that CheD plays an important role in the chemotaxis and pathogenesis ofB. burgdorferi. We propose potential connections between CheD, CheX, and MCPs and discuss how these interactions play critical roles during the infectious life cycle of the spirochete.

scholarly journals Isolation of a gene required for programmed initiation of development by Aspergillus nidulans.

1992 ◽  
Vol 12 (9) ◽  
pp. 3827-3833 ◽  
Author(s):  
T H Adams ◽  
W A Hide ◽  
L N Yager ◽  
B N Lee
Keyword(s):  
Life Cycle ◽  
Aspergillus Nidulans ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Nutrient Deprivation ◽  
Deletion Mutants ◽  
Wild Type ◽  
Microbial Development ◽  
Type Strains ◽  
Posttranscriptional Level

In contrast to many other cases in microbial development, Aspergillus nidulans conidiophore production initiates primarily as a programmed part of the life cycle rather than as a response to nutrient deprivation. Mutations in the acoD locus result in "fluffy" colonies that appear to grow faster than the wild type and proliferate as undifferentiated masses of vegetative cells. We show that unlike wild-type strains, acoD deletion mutants are unable to make conidiophores under optimal growth conditions but can be induced to conidiate when growth is nutritionally limited. The requirement for acoD in conidiophore development occurs prior to activation of brlA, a primary regulator of development. The acoD transcript is present both in vegetative hyphae prior to developmental induction and in developing cultures. However, the effects of acoD mutations are detectable only after developmental induction. We propose that acoD activity is primarily controlled at the posttranscriptional level and that it is required to direct developmentally specific changes that bring about growth inhibition and activation of brlA expression to result in conidiophore development.

scholarly journals Borrelia burgdorferi Lacking BBK32, a Fibronectin-Binding Protein, Retains Full Pathogenicity

2006 ◽  
Vol 74 (6) ◽  
pp. 3305-3313 ◽  
Author(s):  
Xin Li ◽  
Xianzhong Liu ◽  
Deborah S. Beck ◽  
Fred S. Kantor ◽  
Erol Fikrig
Keyword(s):  
Life Cycle ◽  
Borrelia Burgdorferi ◽  
Deletion Mutant ◽  
Binding Protein ◽  
Kanamycin Resistance ◽  
Binding Activity ◽  
Mammalian Host ◽  
Wild Type ◽  
Fibronectin Binding Protein ◽  
Fibronectin Binding

ABSTRACT BBK32, a fibronectin-binding protein of Borrelia burgdorferi, is one of many surface lipoproteins that are differentially expressed by the Lyme disease spirochete at various stages of its life cycle. The level of BBK32 expression in B. burgdorferi is highest during infection of the mammalian host and lowest in flat ticks. This temporal expression profile, along with its fibronectin-binding activity, strongly suggests that BBK32 may play an important role in Lyme pathogenesis in the host. To test this hypothesis, we constructed an isogenic BBK32 deletion mutant from wild-type B. burgdorferi B31 by replacing the BBK32 gene with a kanamycin resistance cassette through homologous recombination. We examined both the wild-type strain and the BBK32 deletion mutant extensively in the experimental mouse-tick model of the Borrelia life cycle. Our data indicated that B. burgdorferi lacking BBK32 retained full pathogenicity in mice, regardless of whether mice were infected artificially by syringe inoculation or naturally by tick bite. The loss of BBK32 expression in the mutant had no adverse effect on spirochete acquisition (mouse-to-tick) and transmission (tick-to-mouse) processes. These results suggest that additional B. burgdorferi proteins can complement the function of BBK32, fibronectin binding or otherwise, during the natural spirochete life cycle.

scholarly journals The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

2015 ◽  
Vol 83 (7) ◽  
pp. 2596-2604 ◽  
Author(s):  
Liyun Liu ◽  
Shuai Hao ◽  
Ruiting Lan ◽  
Guangxia Wang ◽  
Di Xiao ◽  
...  
Keyword(s):  
Secretion System ◽  
Host Cells ◽  
Target Cells ◽  
Deletion Mutants ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Mutant Strains

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells.Citrobacter freundiistrain CF74 has a complete T6SS genomic island (GI) that containsclpV,hcp-2, andvgrT6SS genes. We constructedclpV,hcp-2,vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased inclpV,hcp-2,vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role inC. freundii.

scholarly journals An Ixodes scapularis Protein Disulfide Isomerase Contributes to Borrelia burgdorferi Colonization of the Vector

2020 ◽  
Vol 88 (12) ◽  
Author(s):  
Yongguo Cao ◽  
Connor Rosen ◽  
Gunjan Arora ◽  
Akash Gupta ◽  
Carmen J. Booth ◽  
...  
Keyword(s):  
Life Cycle ◽  
Borrelia Burgdorferi ◽  
Protein Disulfide Isomerase ◽  
Inflammatory Responses ◽  
Ixodes Scapularis ◽  
Vertebrate Host ◽  
Content Type ◽  
Disulfide Isomerase ◽  
Complex Interactions ◽  
Protein Disulfide

ABSTRACT Borrelia burgdorferi causes Lyme disease, the most common tick-transmitted illness in North America. When Ixodes scapularis feed on an infected vertebrate host, spirochetes enter the tick gut along with the bloodmeal and colonize the vector. Here, we show that a secreted tick protein, I. scapularis protein disulfide isomerase A3 (IsPDIA3), enhances B. burgdorferi colonization of the tick gut. I. scapularis ticks in which ispdiA3 has been knocked down using RNA interference have decreased spirochete colonization of the tick gut after engorging on B. burgdorferi-infected mice. Moreover, administration of IsPDIA3 antiserum to B. burgdorferi-infected mice reduced the ability of spirochetes to colonize the tick when feeding on these animals. We show that IsPDIA3 modulates inflammatory responses at the tick bite site, potentially facilitating spirochete survival at the vector-host interface as it exits the vertebrate host to enter the tick gut. These data provide functional insights into the complex interactions between B. burgdorferi and its arthropod vector and suggest additional targets to interfere with the spirochete life cycle.

scholarly journals Oligopeptide Permease A5 Modulates Vertebrate Host-Specific Adaptation of Borrelia burgdorferi

2011 ◽  
Vol 79 (8) ◽  
pp. 3407-3420 ◽  
Author(s):  
B. V. Subba Raju ◽  
Maria D. Esteve-Gassent ◽  
S. L. Rajasekhar Karna ◽  
Christine L. Miller ◽  
Tricia A. Van Laar ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Immunoblot Analysis ◽  
Vertebrate Host ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Content Type ◽  
Specific Adaptation ◽  
Protein Levels ◽  
Oligopeptide Permease

ABSTRACTBorrelia burgdorferi, the agent of Lyme disease, undergoes rapid adaptive gene expression in response to signals unique to its arthropod vector or vertebrate hosts. Among the upregulated genes under vertebrate host conditions is one of the five annotated homologs of oligopeptide permease A (OppA5, BBA34). A mutant lackingoppA5was constructed in an lp25-deficient isolate ofB. burgdorferistrain B31, and the minimal regions of infectivity were restored via a shuttle vector pBBE22 with or without an intact copy ofbba34. Immunoblot analysis of thebba34mutant revealed a reduction in the levels of RpoS, BosR, and CsrABbwith a concomitant reduction in the levels of OspC, DbpA, BBK32, and BBA64. There were no changes in the levels of OspA, NapA, P66, and three other OppA orthologs. Quantitative transcriptional analysis correlated with the changes in the protein levels. However, thebba34mutant displayed comparable infectivities in the C3H/HeN mice and the wild-type strain, despite the reduction in several pathogenesis-related proteins. Supplementation of the growth medium with increased levels of select components, notably sodium acetate and sodium bicarbonate, restored the levels of several proteins in thebba34mutant to wild-type levels. We speculate that the transport of acetate appears to contribute to the accumulation of key metabolites, like acetyl phosphate, that facilitate the adaptation ofB. burgdorferito the vertebrate host by the activation of the Rrp2-RpoN-RpoS pathway. These studies underscore the importance of solute transport to host-specific adaptation ofB. burgdorferi.

scholarly journals Analysis of the HD-GYP Domain Cyclic Dimeric GMP Phosphodiesterase Reveals a Role in Motility and the Enzootic Life Cycle of Borrelia burgdorferi

2011 ◽  
Vol 79 (8) ◽  
pp. 3273-3283 ◽  
Author(s):  
Syed Z. Sultan ◽  
Joshua E. Pitzer ◽  
Tristan Boquoi ◽  
Gerry Hobbs ◽  
Michael R. Miller ◽  
...  
Keyword(s):  
Life Cycle ◽  
Borrelia Burgdorferi ◽  
Double Mutant ◽  
Ixodes Scapularis ◽  
Metabolizing Enzymes ◽  
Content Type ◽  
Motility Regulation ◽  
Mutant Cells ◽  
Single Set

ABSTRACTHD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria.Borrelia burgdorferipossesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation inpdeAresulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with aKmof 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing thepdeA pdeBdouble mutant, we demonstrate that no additional phosphodiesterases are present inB. burgdorferi.pdeBsingle mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing apilZpdeBdouble mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly inpdeBmutant cells, these cells exhibited a reduced ability to survive inIxodes scapularisticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role ofpdeBincreases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle ofB. burgdorferi.

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