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Dive into the research topics where Daniel C. Desrosiers is active.

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Featured researches published by Daniel C. Desrosiers.


Protein Science | 2004

The ankyrin repeat as molecular architecture for protein recognition

Leila K. Mosavi; Tobin J. Cammett; Daniel C. Desrosiers; Zheng-yu Peng

The ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. This protein–protein interaction module is involved in a diverse set of cellular functions, and consequently, defects in ankyrin repeat proteins have been found in a number of human diseases. Recent biophysical, crystallographic, and NMR studies have been used to measure the stability and define the various topological features of this motif in an effort to understand the structural basis of ankyrin repeat‐mediated protein–protein interactions. Characterization of the folding and assembly pathways suggests that ankyrin repeat domains generally undergo a two‐state folding transition despite their modular structure. Also, the large number of available sequences has allowed the ankyrin repeat to be used as a template for consensus‐based protein design. Such projects have been successful in revealing positions responsible for structure and function in the ankyrin repeat as well as creating a potential universal scaffold for molecular recognition.


Molecular Microbiology | 2007

The Lyme disease agent Borrelia burgdorferi requires BB0690, a Dps homologue, to persist within ticks.

Xin Li; Utpal Pal; Nandhini Ramamoorthi; Xianzhong Liu; Daniel C. Desrosiers; Christian H. Eggers; John F. Anderson; Justin D. Radolf; Erol Fikrig

Borrelia burgdorferi survives in an enzootic cycle, and Dps proteins protect DNA against damage during starvation or oxidative stress. The role of a Dps homologue encoded by Borrelia in spirochaete survival was assessed. Dps‐deficient spirochaetes were infectious in mice via needle‐inoculation at the dose of 105 spirochaetes. Larval ticks successfully acquired Dps‐deficient spirochaetes via a blood meal on mice. However, after extended periods within unfed nymphs, the Dps‐deficient spirochaetes failed to be transmitted to a new host when nymphs fed. Our data suggest that Dps functions to protect the spirochaetes during dormancy in unfed ticks, and in its absence, the spirochaetes become susceptible during tick feeding. dps is differentially expressed in vivo– low in mice and high in ticks – but constitutively expressed in vitro, showing little change during growth or in response to oxidative stress. Borrelia Dps forms a dodecameric complex capable of sequestering iron. The Dps‐deficient spirochaetes showed no defect in starvation and oxidative stress assays, perhaps due to the lack of iron in spirochaetes grown in vitro. Dps is critical for spirochaete persistence within ticks, and strategies to interfere with Dps could potentially reduce Borrelia populations in nature and thereby influence the incidence of Lyme disease.


Molecular Microbiology | 2007

The general transition metal (Tro) and Zn2+ (Znu) transporters in Treponema pallidum: analysis of metal specificities and expression profiles

Daniel C. Desrosiers; Yong Cheng Sun; Akbar A. Zaidi; Christian H. Eggers; David L. Cox; Justin D. Radolf

Acquisition of transition metals is central to the struggle between a bacterial pathogen and its mammalian host. Previous studies demonstrated that Treponema pallidum encodes a cluster‐9 (C9) ABC transporter (troABCD) whose solute‐binding protein component (TroA) ligands Zn2+ and Mn2+ with essentially equal affinities. Bioinformatic analysis revealed that T. pallidum encodes an additional C9 transporter (tp0034–36) orthologous to Zn2+‐uptake (Znu) systems in other bacteria; the binding protein component, ZnuA, contains a His‐rich tract characteristic of C9 Zn2+‐binding proteins. Metal analysis and metal‐reconstitution studies demonstrated that ZnuA is a Zn2+‐binding protein; parallel studies confirmed that TroA binds Zn2+, Mn2+ and Fe. Circular dichroism showed that ZnuA, but not TroA, undergoes conformational changes in the presence of Zn2+. Using isothermal titration calorimetry (ITC), we demonstrated that TroA binds Zn2+ and Mn2+ with affinities approximately 100‐fold greater than those previously reported. ITC analysis revealed that ZnuA contains multiple Zn2+‐binding sites, two of which are high‐affinity and presumed to be located within the binding pocket and His‐rich loop. Quantitative reverse transcription polymerase chain reaction of tro and znu transcripts combined with immunoblot analysis of TroA and ZnuA confirmed that both transporters are simultaneously expressed in T. pallidum and that TroA is expressed at much greater levels than ZnuA. Collectively, our findings indicate that T. pallidum procures transition metals via the concerted utilization of its general metal (Tro) and Zn2+ (Znu) transporters. Sequestration of periplasmic Zn2+ by ZnuA may free up TroA binding capacity for the importation of Fe and Mn2+.


Infection and Immunity | 2010

Surface Immunolabeling and Consensus Computational Framework To Identify Candidate Rare Outer Membrane Proteins of Treponema pallidum

David L. Cox; Amit Luthra; Star Dunham-Ems; Daniel C. Desrosiers; Juan C. Salazar; Melissa J. Caimano; Justin D. Radolf

ABSTRACT Treponema pallidum reacts poorly with the antibodies present in rabbit and human syphilitic sera, a property attributed to the paucity of proteins in its outer membrane. To better understand the basis for the syphilis spirochetes “stealth pathogenicity,” we used a dual-label, 3-step amplified assay in which treponemes encapsulated in gel microdroplets were probed with syphilitic sera in parallel with anti-FlaA antibodies. A small (approximately 5 to 10%) but reproducible fraction of intact treponemes bound IgG and/or IgM antibodies. Three lines of evidence supported the notion that the surface antigens were likely β-barrel-forming outer membrane proteins (OMPs): (i) surface labeling with anti-lipoidal (VDRL) antibodies was not observed, (ii) immunoblot analysis confirmed prior results showing that T. pallidum glycolipids are not immunoreactive, and (iii) labeling of intact organisms was not appreciably affected by proteinase K (PK) treatment. With this method, we also demonstrate that TprK (TP0897), an extensively studied candidate OMP, and TP0136, a lipoprotein recently reported to be surface exposed, are both periplasmic. Consistent with the immunolabeling studies, TprK was also found to lack amphiphilicity, a characteristic property of β-barrel-forming proteins. Using a consensus computational framework that combined subcellular localization and β-barrel structural prediction tools, we generated ranked groups of candidate rare OMPs, the predicted T. pallidum outer membrane proteome (OMPeome), which we postulate includes the surface-exposed molecules detected by our enhanced gel microdroplet assay. In addition to underscoring the syphilis spirochetes remarkably poor surface antigenicity, our findings help to explain the complex and shifting balance between pathogen and host defenses that characterizes syphilitic infection.


Infection and Immunity | 2011

The Hybrid Histidine Kinase Hk1 Is Part of a Two-Component System That Is Essential for Survival of Borrelia burgdorferi in Feeding Ixodes scapularis Ticks

Melissa J. Caimano; Melisha R. Kenedy; Toru Kairu; Daniel C. Desrosiers; Michael W. Harman; Star Dunham-Ems; Darrin R. Akins; Utpal Pal; Justin D. Radolf

ABSTRACT Two-component systems (TCS) are principal mechanisms by which bacteria adapt to their surroundings. Borrelia burgdorferi encodes only two TCS. One is comprised of a histidine kinase, Hk2, and the response regulator Rrp2. While the contribution of Hk2 remains unclear, Rrp2 is part of a regulatory pathway involving the spirochetes alternate sigma factors, RpoN and RpoS. Genes within the Rrp2/RpoN/RpoS regulon function to promote tick transmission and early infection. The other TCS consists of a hybrid histidine kinase, Hk1, and the response regulator Rrp1. Hk1 is composed of two periplasmic sensor domains (D1 and D2), followed by conserved cytoplasmic histidine kinase core, REC, and Hpt domains. In addition to its REC domain, Rrp1 contains a GGDEF motif characteristic of diguanylate cyclases. To investigate the role of Hk1 during the enzootic cycle, we inactivated this gene in two virulent backgrounds. Extensive characterization of the resulting mutants revealed a dramatic phenotype whereby Hk1-deficient spirochetes are virulent in mice and able to migrate out of the bite site during feeding but are killed within the midgut following acquisition. We hypothesize that the phosphorelay between Hk1 and Rrp1 is initiated by the binding of feeding-specific ligand(s) to Hk1 sensor domain D1 and/or D2. Once activated, Rrp1 directs the synthesis of cyclic dimeric GMP (c-di-GMP), which, in turn, modulates the expression and/or activity of gene products required for survival within feeding ticks. In contrast to the Rrp2/RpoN/RpoS pathway, which is active only within feeding nymphs, the Hk1/Rrp1 TCS is essential for survival during both larval and nymphal blood meals.


Infection and Immunity | 2010

Znu is the predominant zinc importer in Yersinia pestis during in vitro growth but is not essential for virulence.

Daniel C. Desrosiers; Scott W. Bearden; Ildefonso Mier; Jennifer Abney; James T. Paulley; Jacqueline D. Fetherston; Juan C. Salazar; Justin D. Radolf; Robert D. Perry

ABSTRACT Little is known about Zn homeostasis in Yersinia pestis, the plague bacillus. The Znu ABC transporter is essential for zinc (Zn) uptake and virulence in a number of bacterial pathogens. Bioinformatics analysis identified ZnuABC as the only apparent high-affinity Zn uptake system in Y. pestis. Mutation of znuACB caused a growth defect in Chelex-100-treated PMH2 growth medium, which was alleviated by supplementation with submicromolar concentrations of Zn. Use of transcriptional reporters confirmed that Zur mediated Zn-dependent repression and that it can repress gene expression in response to Zn even in the absence of Znu. Virulence testing in mouse models of bubonic and pneumonic plague found only a modest increase in survival in low-dose infections by the znuACB mutant. Previous studies of cluster 9 (C9) transporters suggested that Yfe, a well-characterized C9 importer for manganese (Mn) and iron in Y. pestis, might function as a second, high-affinity Zn uptake system. Isothermal titration calorimetry revealed that YfeA, the solute-binding protein component of Yfe, binds Mn and Zn with comparably high affinities (dissociation constants of 17.8 ± 4.4 nM and 6.6 ± 1.2 nM, respectively), although the complete Yfe transporter could not compensate for the loss of Znu in in vitro growth studies. Unexpectedly, overexpression of Yfe interfered with the znu mutants ability to grow in low concentrations of Zn, while excess Zn interfered with the ability of Yfe to import iron at low concentrations; these results suggest that YfeA can bind Zn in the bacterial cell but that Yfe is incompetent for transport of the metal. In addition to Yfe, we have now eliminated MntH, FetMP, Efe, Feo, a substrate-binding protein, and a putative nickel transporter as the unidentified, secondary Zn transporter in Y. pestis. Unlike other bacterial pathogens, Y. pestis does not require Znu for high-level infectivity and virulence; instead, it appears to possess a novel class of transporter, which can satisfy the bacteriums Zn requirements under in vivo metal-limiting conditions. Our studies also underscore the need for bacterial cells to balance binding and transporter specificities within the periplasm in order to maintain transition metal homeostasis.


Molecular Microbiology | 2011

TP0326, a Treponema pallidum β-barrel assembly machinery A (BamA) orthologue and rare outer membrane protein.

Daniel C. Desrosiers; Arvind Anand; Amit Luthra; Star Dunham-Ems; Morgan LeDoyt; Michael A. D. Cummings; Azad Eshghi; Adriana R. Cruz; Juan C. Salazar; Melissa J. Caimano; Justin D. Radolf

Definitive identification of Treponema pallidum rare outer membrane proteins (OMPs) has long eluded researchers. TP0326, the sole protein in T. pallidum with sequence homology to a Gram‐negative OMP, belongs to the BamA family of proteins essential for OM biogenesis. Structural modelling predicted that five polypeptide transport‐associated (POTRA) domains comprise the N‐terminus of TP0326, while the C‐terminus forms an 18‐stranded amphipathic β‐barrel. Circular dichroism, heat modifiability by SDS‐PAGE, Triton X‐114 phase partitioning and liposome incorporation supported these topological predictions and confirmed that the β‐barrel is responsible for the native proteins amphiphilicity. Expression analyses revealed that native TP0326 is expressed at low abundance, while a protease‐surface accessibility assay confirmed surface exposure. Size‐exclusion chromatography and blue native polyacrylamide gel electrophoresis revealed a modular Bam complex in T. pallidum larger than that of Escherichia coli. Non‐orthologous ancillary factors and self‐association of TP0326 via its β‐barrel may both contribute to the Bam complex. T. pallidum‐infected rabbits mount a vigorous antibody response to both POTRA and β‐barrel portions of TP0326, whereas humans with secondary syphilis respond predominantly to POTRA. The syphilis spirochaete appears to have devised a stratagem for harnessing the Bam pathway while satisfying its need to limit surface antigenicity.


Molecular Microbiology | 2010

Identification of residues within ligand-binding domain 1 (LBD1) of the Borrelia burgdorferi OspC protein required for function in the mammalian environment.

Christopher G. Earnhart; DeLacy V. LeBlanc; Katie E. Alix; Daniel C. Desrosiers; Justin D. Radolf; Richard T. Marconi

Borrelia burgdorferi outer surface protein C (ospC) is required for the establishment of infection in mammals. However, its precise function remains controversial. The biologically active form of OspC appears to be a homodimer. Alpha helix 1 and 1′ of the apposing monomers form a solvent‐accessible pocket at the dimeric interface that presents a putative ligand‐binding domain (LBD1). Here we employ site‐directed and allelic‐exchange mutagenesis to test the hypothesis that LBD1 is a determinant of OspC function in the mammalian environment. Substitution of residues K60, E61 and E63 which line LBD1 resulted in the loss of infectivity or influenced dissemination. Analyses of the corresponding recombinant proteins demonstrated that the loss of function was not due to structural perturbation, impaired dimer formation or the loss of plasminogen binding. This study is the first to assess the involvement of individual residues and domains of OspC in its in vivo function. The data support the hypothesis that OspC interacts with a mammalian derived ligand that is critical for survival during early infection. These results shed new light on the structure–functions relationships of OspC and challenge existing hypotheses regarding OspC function in mammals.


Journal of Bacteriology | 2005

Tp0453, A Concealed Outer Membrane Protein Of Treponema Pallidum, Enhances Membrane Permeability

Karsten R. O. Hazlett; David L. Cox; Marc Decaffmeyer; Michael P. Bennett; Daniel C. Desrosiers; Carson J. La Vake; Morgan E. La Vake; Kenneth W. Bourell; Esther J. Robinson; Robert Brasseur; Justin D. Radolf

The outer membrane of Treponema pallidum, the non-cultivable agent of venereal syphilis, contains a paucity of protein(s) which has yet to be definitively identified. In contrast, the outer membranes of gram-negative bacteria contain abundant immunogenic membrane-spanning beta-barrel proteins mainly involved in nutrient transport. The absence of orthologs of gram-negative porins and outer membrane nutrient-specific transporters in the T. pallidum genome predicts that nutrient transport across the outer membrane must differ fundamentally in T. pallidum and gram-negative bacteria. Here we describe a T. pallidum outer membrane protein (TP0453) that, in contrast to all integral outer membrane proteins of known structure, lacks extensive beta-sheet structure and does not traverse the outer membrane to become surface exposed. TP0453 is a lipoprotein with an amphiphilic polypeptide containing multiple membrane-inserting, amphipathic alpha-helices. Insertion of the recombinant, non-lipidated protein into artificial membranes results in bilayer destabilization and enhanced permeability. Our findings lead us to hypothesize that TP0453 is a novel type of bacterial outer membrane protein which may render the T. pallidum outer membrane permeable to nutrients while remaining inaccessible to antibody.


Journal of Bacteriology | 2007

Borrelia burgdorferi BBA74, a Periplasmic Protein Associated with the Outer Membrane, Lacks Porin-Like Properties

Vishwaroop Mulay; Melissa J. Caimano; Dionysios Liveris; Daniel C. Desrosiers; Justin D. Radolf; Ira Schwartz

The outer membrane of Borrelia burgdorferi, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% alpha-helix with little beta-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins.

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Justin D. Radolf

University of Connecticut Health Center

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Melissa J. Caimano

University of Connecticut Health Center

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David L. Cox

Centers for Disease Control and Prevention

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Star Dunham-Ems

University of Connecticut Health Center

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Amit Luthra

University of Connecticut Health Center

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Juan C. Salazar

University of Connecticut

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Arvind Anand

University of Connecticut Health Center

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Carson J. La Vake

University of Connecticut Health Center

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