Summary
Systemic dissemination of microbial pathogens permits microbes to spread from the initial site of infection to secondary target tissues and is responsible for most mortality due to bacterial ...infections. Dissemination is a critical stage of disease progression by the Lyme spirochaete, Borrelia burgdorferi. However, many mechanistic features of the process are not yet understood. A key step is adhesion of circulating microbes to vascular surfaces in the face of the shear forces present in flowing blood. Using real‐time microscopic imaging of the Lyme spirochaete in living mice we previously identified the first bacterial protein (B. burgdorferi BBK32) shown to mediate vascular adhesion in vivo. Vascular adhesion is also dependent on host fibronectin (Fn) and glycosaminoglycans (GAGs). In the present study, we investigated the mechanisms of BBK32‐dependent vascular adhesion in vivo. We determined that BBK32–Fn interactions (tethering) function as a molecular braking mechanism that permits the formation of more stable BBK32–GAG interactions (dragging) between circulating bacteria and vascular surfaces. Since BBK32‐like proteins are expressed in a variety of pathogens we believe that the vascular adhesion mechanisms we have deciphered here may be critical for understanding the dissemination mechanisms of other bacterial pathogens.
Significance Invariant natural killer T cells (iNKT) have been found primarily patrolling inside blood vessels in the liver, where they respond to bacterial glycolipids presented by CD1d on liver ...macrophages. We show joint iNKT cells are localized outside of blood vessels and respond directly to the joint-homing pathogen, Borrelia burgdorferi , which causes Lyme borreliosis using multichannel spinning-disk intravital microscopy. These iNKT cells interacted with B. burgdorferi at the vessel wall and disrupted its dissemination attempts into joints. Successful penetrance of B. burgdorferi out of the vasculature and into the joint tissue was met by a lethal attack by extravascular iNKT cells through a granzyme-dependent pathway. These results suggest a critical extravascular iNKT cell immune surveillance in joints that functions as a cytotoxic barrier.
CXCR6-GFP ⁺ cells, which encompass 70% invariant natural killer T cells (iNKT cells), have been found primarily patrolling inside blood vessels in the liver. Although the iNKT cells fail to interact with live pathogens, they do respond to bacterial glycolipids presented by CD1d on liver macrophage that have caught the microbe. In contrast, in this study using dual laser multichannel spinning-disk intravital microscopy of joints, the CXCR6-GFP, which also made up 60–70% iNKT cells, were not found in the vasculature but rather closely apposed to and surrounding the outside of blood vessels, and to a lesser extent throughout the extravascular space. These iNKT cells also differed in behavior, responding rapidly and directly to joint-homing pathogens like Borrelia burgdorferi , which causes Lyme disease. These iNKT cells interacted with B. burgdorferi at the vessel wall and disrupted dissemination attempts by these microbes into joints. Successful penetrance of B. burgdorferi out of the vasculature and into the joint tissue was met by a lethal attack by extravascular iNKT cells through a granzyme-dependent pathway, an observation also made in vitro for iNKT cells from joint but not liver or spleen. These results suggest a novel, critical extravascular iNKT cell immune surveillance in joints that functions as a cytotoxic barrier and explains a large increase in pathogen burden of B. burgdorferi in the joint of iNKT cell-deficient mice, and perhaps the greater susceptibility of humans to this pathogen because of fewer iNKT cells in human joints.
Spirochetes causing Lyme borreliosis are obligate parasites that can only be found in a tick vector or a vertebrate host. The ability to survive in these two disparate environments requires up and ...downregulation of specific genes by regulatory circuits that remain largely obscure. In this work on the Lyme spirochete, B. burgdorferi, we show that a disruption of the hrpA gene, which encodes a putative RNA helicase, results in a complete loss in the ability of the spirochetes to infect mice by needle inoculation. Studies of protein expression in culture by 2D gels revealed a change in the expression of 33 proteins in hrpA clones relative to the wild-type parent. Quantitative characterization of protein expression by iTRAQ analysis revealed a total of 187 differentially regulated proteins in an hrpA background: 90 downregulated and 97 upregulated. Forty-two of the 90 downregulated and 65 of the 97 upregulated proteins are not regulated under any conditions by the previously reported regulators in B. burgdorferi (bosR, rrp2, rpoN, rpoS or rrp1). Downregulated and upregulated proteins also fell into distinct functional categories. We conclude that HrpA is part of a new and distinct global regulatory pathway in B. burgdorferi gene expression. Because an HrpA orthologue is present in many bacteria, its participation in global regulation in B. burgdorferi may have relevance in other bacterial species where its function remains obscure. We believe this to be the first report of a role for an RNA helicase in a global regulatory pathway in bacteria. This finding is particularly timely with the recent growth of the field of RNA regulation of gene expression and the ability of RNA helicases to modulate RNA structure and function.
The Lyme disease spirochete Borrelia burgdorferi must differentially express genes and proteins in order to survive in and transit between its tick vector and vertebrate reservoir. The putative ...DEAH-box RNA helicase, HrpA, has been recently identified as an addition to the spirochete's global regulatory machinery; using proteomic methods, we demonstrated that HrpA modulates the expression of at least 180 proteins. Although most bacteria encode an HrpA helicase, RNA helicase activity has never been demonstrated for HrpAs and the literature contains little information on the contribution of this protein to bacterial physiology or pathogenicity. In this work, we report that B. burgdorferi HrpA has RNA-stimulated ATPase activity and RNA helicase activity and that this enzyme is essential for both mammalian infectivity by syringe inoculation and tick transmission. Reduced infectivity of strains carrying mutations in the ATPase and RNA binding motif mutants suggests that full virulence expression requires both ATPase and coupled helicase activity. Microarray profiling revealed changes in RNA levels of two-fold, or less in an hrpA mutant versus wild-type, suggesting that the enzyme functions largely or exclusively at the post-transcriptional level. In this regard, northern blot analysis of selected gene products highly regulated by HrpA (bb0603 p66, bba74, bb0241 glpK, bb0242 and bb0243 glpA) suggests a role for HrpA in the processing and translation of transcripts. In addition to being the first demonstration of RNA helicase activity for a bacterial HrpA, our data indicate that the post-transcriptional regulatory functions of this enzyme are essential for maintenance of the Lyme disease spirochete's enzootic cycle.
Borrelia species are unique in the bacterial world in possessing segmented genomes which sometimes contain over 20 genetic elements. Most elements are linear and contain covalently closed hairpin ...ends requiring a specialized process, telomere resolution, for their generation. Hairpin telomere resolution is mediated by the telomere resolvase, ResT. Although the process has been studied extensively in vitro, the essential nature of the resT gene has precluded biological studies to further probe the role of ResT. In this work, we have generated a B. burgdorferi strain that carries an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible resT gene controlled by a tightly regulated promoter. ResT is expressed in this strain at ∼14,000 monomers per cell, similar to the ∼15,000 monomers observed for the parental strain. We demonstrate ResT depletion with a half-life of 16 h upon IPTG washout. ResT depletion resulted in arrested growth 48 h after washout. Interestingly, not all spirochetes died after ResT washout, and at least 15% remained quiescent and could be resuscitated even at 2 weeks postwashout. Significant levels of DNA synthesis were not observed upon growth arrest, suggesting that ResT might interact directly or indirectly with factors controlling the initiation or elongation of DNA synthesis. Analysis of the linear plasmids lp17 and lp28-2 showed that the linear forms of these plasmids began to disappear and be replaced by higher-molecular-weight forms by 24 h post-IPTG washout. Treatment of DNA from the ResT-depleted strain with ResT in vitro revealed the presence of replicated telomeres expected in replication intermediates.
The Lyme disease spirochete Borrelia burgdorferi must differentially express genes and proteins in order to survive in and transit between its tick vector and vertebrate reservoir. The putative ...DEAH-box RNA helicase, HrpA, has been recently identified as an addition to the spirochete's global regulatory machinery; using proteomic methods, we demonstrated that HrpA modulates the expression of at least 180 proteins. Although most bacteria encode an HrpA helicase, RNA helicase activity has never been demonstrated for HrpAs and the literature contains little information on the contribution of this protein to bacterial physiology or pathogenicity. In this work, we report that B. burgdorferi HrpA has RNA-stimulated ATPase activity and RNA helicase activity and that this enzyme is essential for both mammalian infectivity by syringe inoculation and tick transmission. Reduced infectivity of strains carrying mutations in the ATPase and RNA binding motif mutants suggests that full virulence expression requires both ATPase and coupled helicase activity. Microarray profiling revealed changes in RNA levels of two-fold, or less in an hrpA mutant versus wild-type, suggesting that the enzyme functions largely or exclusively at the post-transcriptional level. In this regard, northern blot analysis of selected gene products highly regulated by HrpA (bb0603 p66, bba74, bb0241 glpK, bb0242 and bb0243 glpA) suggests a role for HrpA in the processing and translation of transcripts. In addition to being the first demonstration of RNA helicase activity for a bacterial HrpA, our data indicate that the post-transcriptional regulatory functions of this enzyme are essential for maintenance of the Lyme disease spirochete's enzootic cycle.
We have conjugated bovine serum albumin (BSA) to poly(
N-isopropylacrylamide-co-acrylic acid) (poly(NIPAAm-AA)) by using water-soluble carbodiimide, and the effects of the bulk mass ratio of protein ...to polymer (
r) on the formation of polymer–protein conjugates have been studied. HPLC, electrophoresis, viscosimetry and fluorescence spectroscopy suggest that the mode of covalent binding of BSA to poly(NIPAAm-AA) depends upon the weight concentration ratio (
r) of BSA to poly(NIPAAm-AA). At
r⩽1, free poly(NIPAAm-AA) molecules coexist with conjugate, and when
r reaches 1 the amount of free polymer is too small to be observed. It is shown that depending on the ratio
r, two types of conjugate particles were formed: at
r<1, the protein molecules in the structure of conjugate particles are densely covered as a shell by polymer chain and practically “fenced off” from water environment; at
r>1 the conjugate-forming particles possess more friable structures in which protein molecules are practically exposed to the solvent. The complex formation involving electrostatic interactions between BSA and carbodiimide activated polymer are proposed as the driving force for the covalent binding of BSA to polymer macromolecules. The coil-globule transition of macromolecules in low and thermally induced precipitation in more concentrated solutions of bioconjugates was observed. The immunogenic properties of covalent conjugates of CP–BSA were investigated and the temperature-modulated solubility–immunogenicity alterations was analyzed. A single immunization of mice with conjugates at the thermally precipitating concentration without an adjuvant evoked increased specific immune response to BSA, which practically did not depend on the initial conjugation ratio of components. Such a modulated system is attractive for application as a novel immunogenic system in vaccine technology.
The emerging need for rapid screening and identification methods for microbiological purposes necessitates the combined uses of high-tech instruments. In this work, electrospray ionization mass ...spectrometry was used to visualize the relation of ten newly isolated moderately halophilic microorganisms, to Halomonas salina DSMZ 5928 and Halomonas halophila DSMZ 4770. The method was based on the global analysis of the metabolites in culture media and is termed as metabolic footprinting. Since it was not possible to gain insight into the similarities solely based on the visual inspection of the chromatograms, principal component (PC) analysis was applied on the data. Three PCs alone were able to explain 99% of the information in the data set. The score plots revealed the relation of the new isolates to the two type strains whereas the loading plots gave important clues on the significant ions responsible for the observed clustering. Loading plots also indicated inversely correlated ions that give clues on differing metabolic pathways. The work described here offers a potentially useful way for preliminary rapid phenotypic characterization of new and closely related isolates and a method for screening of similar microorganisms for different and valuable secondary metabolites.
PDF
