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 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 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 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 Genetic Manipulations of the Hyperthermophilic Piezophilic Archaeon Thermococcus barophilus

2014 ◽  
Vol 80 (7) ◽  
pp. 2299-2306 ◽  
Author(s):  
Axel Thiel ◽  
Grégoire Michoud ◽  
Yann Moalic ◽  
Didier Flament ◽  
Mohamed Jebbar
Keyword(s):  
Gene Disruption ◽  
Negative Selection ◽  
Deletion Mutants ◽  
Wild Type ◽  
Genetic Studies ◽  
Content Type ◽  
Genetic Manipulations ◽  
Starting Point ◽  
Flanking Regions ◽  
Markerless Deletion

ABSTRACTIn this study, we developed a gene disruption system forThermococcus barophilususing simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single- and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-typeT. barophilusMP cells. Initially, apyrFdeletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of thehisBgene was also constructed in the UBOCC-3256 (ΔpyrF) background, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functionalpyrForhisBallele fromT. barophiluswas inserted into the chromosome of UBOCC-3256 (ΔpyrF) or UBOCC-3260 (ΔpyrFΔhisB), allowing homologous complementation of these mutants. The piezophilic genetic tools developed in this study provide a way to construct strains with multiple genetic backgrounds that will allow further genetic studies for hyperthermophilic piezophilic archaea.

scholarly journals Ergothioneine Is a Secreted Antioxidant in Mycobacterium smegmatis

2013 ◽  
Vol 57 (7) ◽  
pp. 3202-3207 ◽  
Author(s):  
Carine Sao Emani ◽  
Monique J. Williams ◽  
Ian J. Wiid ◽  
Nicholas F. Hiten ◽  
Albertus J. Viljoen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mycobacterium Smegmatis ◽  
Double Mutant ◽  
Culture Media ◽  
Growth Cycle ◽  
Low Molecular Weight ◽  
Deficient Mutant ◽  
Wild Type ◽  
Content Type

ABSTRACTErgothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle ofMycobacterium smegmatisrevealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant ofM. smegmatislackingegtD(MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion ofegtDfrom wild-typeM. smegmatisand an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG- and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protectingM. smegmatisagainst oxidative stress and that ERG is able to partly compensate for the loss of MSH.

scholarly journals Determinants of Intrinsic Aminoglycoside Resistance in Pseudomonas aeruginosa

2012 ◽  
Vol 56 (11) ◽  
pp. 5591-5602 ◽  
Author(s):  
Thomas Krahn ◽  
Christie Gilmour ◽  
Justin Tilak ◽  
Sebastien Fraud ◽  
Nicholas Kerr ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Envelope ◽  
Acquired Resistance ◽  
Insertion Mutant ◽  
Deletion Mutants ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Single Deletion ◽  
High Level ◽  
Increased Susceptibility

ABSTRACTScreening of a transposon insertion mutant library ofPseudomonas aeruginosafor increased susceptibility to paromomycin identified a number of genes whose disruption enhanced susceptibility of this organism to multiple aminoglycosides, including tobramycin, amikacin, and gentamicin. These included genes associated with lipid biosynthesis or metabolism (lptA,faoA), phosphate uptake (pstB), and two-component regulators (amgRS, PA2797-PA2798) and a gene of unknown function (PA0392). Deletion mutants lacking these showed enhanced panaminoglycoside susceptibility that was reversed by the cloned genes, confirming their contribution to intrinsic panaminoglycoside resistance. None of these mutants showed increased aminoglycoside permeation of the cell envelope, indicating that increased susceptibility was not related to enhanced aminoglycoside uptake owing to a reduced envelope barrier function. Several mutants (pstB,faoA, PA0392,amgR) did, however, show increased cytoplasmic membrane depolarization relative to wild type following gentamicin exposure, consistent with the membranes of these mutants being more prone to perturbation, likely by gentamicin-generated mistranslated polypeptides. Mutants lacking any two of these resistance genes in various combinations invariably showed increased aminoglycoside susceptibility relative to single-deletion mutants, confirming their independent contribution to resistance and highlighting the complexity of the intrinsic aminoglycoside resistome inP. aeruginosa. Deletion of these genes also compromised the high-level panaminoglycoside resistance of clinical isolates, emphasizing their important contribution to acquired resistance.

scholarly journals Identification of themcpAandmcpMGenes, Encoding Methyl-Accepting Proteins Involved in Amino Acid and l-Malate Chemotaxis, and Involvement of McpM-Mediated Chemotaxis in Plant Infection by Ralstonia pseudosolanacearum (Formerly Ralstonia solanacearum Phylotypes I and III)

2015 ◽  
Vol 81 (21) ◽  
pp. 7420-7430 ◽  
Author(s):  
Akiko Hida ◽  
Shota Oku ◽  
Takeru Kawasaki ◽  
Yutaka Nakashimada ◽  
Takahisa Tajima ◽  
...  
Keyword(s):  
Amino Acids ◽  
Gene Deletion ◽  
Pcr Analysis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Complete Collection ◽  
Content Type ◽  
Plant Infection ◽  
Tomato Roots ◽  
Ralstonia Pseudosolanacearum

ABSTRACTSequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein (mcp) genes in theRalstonia pseudosolanacearumGMI1000 genome. PCR analysis and DNA sequencing showed that the highly motileR. pseudosolanacearumstrain Ps29 possesses homologs of all 22R. pseudosolanacearumGMI1000mcpgenes. We constructed a complete collection of singlemcpgene deletion mutants ofR. pseudosolanacearumPs29 by unmarked gene deletion. Screening of the mutant collection revealed thatR. pseudosolanacearumPs29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis tol-malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designatedmcpMandmcpA. While wild-typeR. pseudosolanacearumstrain Ps29 displayed attraction to 16 amino acids, themcpAmutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructedmcpAandmcpMdeletion mutants of highly virulentR. pseudosolanacearumstrain MAFF106611 to investigate the contribution of chemotaxis tol-malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, themcpMmutant (but not themcpAmutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis tol-malate, facilitatesR. pseudosolanacearummotility to tomato roots in sand.

scholarly journals The three NADH dehydrogenases of Pseudomonas aeruginosa: Their roles in energy metabolism and links to virulence

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0244142
Author(s):  
Teri N. Hreha ◽  
Sara Foreman ◽  
Ana Duran-Pinedo ◽  
Andrew R. Morris ◽  
Patricia Diaz-Rodriguez ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Opportunistic Pathogen ◽  
Lag Phase ◽  
Deletion Mutants ◽  
Wild Type ◽  
Nadh:Quinone Oxidoreductase ◽  
Double Deletion ◽  
Single Deletion ◽  
Nadh Dehydrogenases ◽  
Pyocyanin Production

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen which relies on a highly adaptable metabolism to achieve broad pathogenesis. In one example of this flexibility, to catalyze the NADH:quinone oxidoreductase step of the respiratory chain, P. aeruginosa has three different enzymes: NUO, NQR and NDH2, all of which carry out the same redox function but have different energy conservation and ion transport properties. In order to better understand the roles of these enzymes, we constructed two series of mutants: (i) three single deletion mutants, each of which lacks one NADH dehydrogenase and (ii) three double deletion mutants, each of which retains only one of the three enzymes. All of the mutants grew approximately as well as wild type, when tested in rich and minimal medium and in a range of pH and [Na+] conditions, except that the strain with only NUO (ΔnqrFΔndh) has an extended lag phase. During exponential phase, the NADH dehydrogenases contribute to total wild-type activity in the following order: NQR > NDH2 > NUO. Some mutants, including the strain without NQR (ΔnqrF) had increased biofilm formation, pyocyanin production, and killed more efficiently in both macrophage and mouse infection models. Consistent with this, ΔnqrF showed increased transcription of genes involved in pyocyanin production.

scholarly journals PFP1, a Gene Encoding an Epc-N Domain-Containing Protein, Is Essential for Pathogenicity of the Barley Pathogen Rhynchosporium commune

2014 ◽  
Vol 13 (8) ◽  
pp. 1026-1035 ◽  
Author(s):  
Sylvia Siersleben ◽  
Daniel Penselin ◽  
Claudia Wenzel ◽  
Sylvie Albert ◽  
Wolfgang Knogge
Keyword(s):  
Upstream Region ◽  
Insertion Mutagenesis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Homologous Proteins ◽  
Gene Encoding ◽  
Fungal Virulence ◽  
Content Type ◽  
Wild Type Level ◽  
Type Gene

ABSTRACTScald caused byRhynchosporium communeis an important foliar disease of barley. Insertion mutagenesis ofR. communegenerated a nonpathogenic fungal mutant which carries the inserted plasmid in the upstream region of a gene namedPFP1. The characteristic feature of the gene product is an Epc-N domain. This motif is also found in homologous proteins shown to be components of histone acetyltransferase (HAT) complexes of fungi and animals. Therefore, PFP1 is suggested to be the subunit of a HAT complex inR. communewith an essential role in the epigenetic control of fungal pathogenicity. TargetedPFP1disruption also yielded nonpathogenic mutants which showed wild-type-like growthex planta, except for the occurrence of hyphal swellings. Complementation of the deletion mutants with the wild-type gene reestablished pathogenicity and suppressed the hyphal swellings. However, despite wild-type-levelPFP1expression, the complementation mutants did not reach wild-type-level virulence. This indicates that the function of the protein complex and, thus, fungal virulence are influenced by a position-affected long-range control ofPFP1expression.

scholarly journals Lysine-specific gingipain K and heme/hemoglobin receptor HmuR are involved in heme utilization in Porphyromonas gingivalis.

2004 ◽  
Vol 51 (1) ◽  
pp. 253-262 ◽  
Author(s):  
Waltena Simpson ◽  
Teresa Olczak ◽  
Caroline A Genco
Keyword(s):  
Porphyromonas Gingivalis ◽  
Type Strain ◽  
Double Mutant ◽  
Wild Type Strain ◽  
Wild Type ◽  
Outer Membrane Receptor ◽  
Iron Source ◽  
Identification And Characterization

We have previously reported on the identification and characterization of the Porphyromonas gingivalis A7436 strain outer membrane receptor HmuR, which is involved in the acquisition of hemin and hemoglobin. We demonstrated that HmuR interacts with the lysine- (Kgp) and arginine- (HRgpA) specific proteases (gingipains) and that Kgp and HRgpA can bind and degrade hemoglobin. Here, we report on the physiological significance of the HmuR-Kgp complex in heme utilization in P. gingivalis through the construction and characterization of a defined kgp mutant and a hmuR kgp double mutant in P. gingivalis A7436. The P. gingivalis kgp mutant exhibited a decreased ability to bind both hemin and hemoglobin. Growth of this strain with hemoglobin was delayed and its ability to utilize hemin as a sole iron source was diminished as compared to the wild type strain. Inactivation of both the hmuR and kgp genes resulted in further decreased ability of P. gingivalis to bind hemoglobin and hemin, as well as diminished ability to utilize either hemin or hemoglobin as a sole iron source. Collectively, these in vivo results further confirmed that both HmuR and Kgp are involved in the utilization of hemin and hemoglobin in P. gingivalis A7436.

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