An important determinant of disease following Streptococcus pneumoniae (pneumococcus) lung infect... more An important determinant of disease following Streptococcus pneumoniae (pneumococcus) lung infection is pulmonary inflammation mediated by polymorphonuclear leukocytes (PMNs). We found that upon intratracheal challenge of mice, recruitment of PMNs into the lungs within the first 3 hours coincided with decreased pulmonary pneumococci, whereas large numbers of pulmonary PMNs beyond 12 hours correlated with a greater bacterial burden. Indeed, mice that survived infection largely resolved inflammation by 72 hours, and PMN depletion at peak infiltration, i.e. 18 hours post-infection, lowered bacterial numbers and enhanced survival. We investigated host signaling pathways that influence both pneumococcus clearance and pulmonary inflammation. Pharmacologic inhibition and/or genetic ablation of enzymes that generate extracellular adenosine (EAD) (e.g. the ectoenzyme CD73) or degrade EAD (e.g. adenosine deaminase) revealed that EAD dramatically increases murine resistance to S. pneumoniae lu...
Medical science monitor: international medical journal of experimental and clinical research
Gait analysis techniques guide the use and design of functional electrical stimulation (FES) syst... more Gait analysis techniques guide the use and design of functional electrical stimulation (FES) systems for paraplegic walking. However, published studies on dynamic gait stability for the effective use of FES are limited. This paper introduces a new risk-tendency graph (RTG) technique to analyze and process gait stability in FES-assisted paraplegic walking. The main instrument was a specialized walker dynamometer system based on a multi-channel strain-gauge bridge network affixed to the frame of the walker. This system collects force information for the handle reaction vector (HRV) between the patient's upper extremities and the walker during walking. The information is then converted into a walker tipping index (WTI), which is an indicator of the patient's walking stability. Dynamic gait stability is then combined with spatio-temporal locating methods for WTI and visually described as morphological curves in the temporal and spatial domains, namely RTGs. To demonstrate the po...
This study evaluated the biomechanical and electromyographic effects of conventional ankle foot o... more This study evaluated the biomechanical and electromyographic effects of conventional ankle foot orthoses (AFOs) and dynamic ankle foot orthoses (DAFOs) on gait in patients with spastic cerebral palsy (CP). Thirteen patients with dynamic equinus underwent motion analysis with electromyography. Both AFOs and DAFOs provided longer stride length, permitted pre-positioning for initial contact, and successfully controlled the excessive plantarflexion during the swing phase. Median frequency (MF) of EMG signal indicated that extremely high firing was found in the patient's lower limbs compared to controls that resulted in tiredness. The DAFOs allowed a significantly larger total ankle range of motion than the AFOs. However, AFOs significantly reduced the MF while DAFOs did not. The reduced MF seen when wearing AFOs suggested an improvement of walking endurance. The DAFO had the advantage of less restriction on ankle movement, which avoids muscular atrophy and improves orthotic compliance. #
Borrelia burgdorferi surface-located membrane protein 1, also known as Lmp1, has been shown to pl... more Borrelia burgdorferi surface-located membrane protein 1, also known as Lmp1, has been shown to play critical roles in pathogen evasion of host-acquired immune defences, thereby facilitating persistent infection. Lmp1 possesses three regions representing potentially discrete domains: Lmp1N, Lmp1M and Lmp1C. Because of its insignificant homology to known proteins, how Lmp1 or its specific regions contribute to microbial biology and infection remains enigmatic. Here, we show that distinct from Lmp1N and Lmp1C, Lmp1M is composed of at least 70% alpha helices and completely lacks recognizable beta sheets. The region binds to host glycosaminoglycan chondroitin-6-sulfate molecules and facilitates mammalian cell attachment, suggesting an adhesin function of Lmp1M. Phenotypic analysis of the Lmp1-deficient mutant engineered to produce Lmp1M on the microbial surface suggests that Lmp1M can independently support B. burgdorferi infectivity in murine hosts. Further exploration of functions of Lmp1 distinct regions will shed new light on the intriguing biology and infectivity of spirochetes and help develop novel interventions to combat Lyme disease.
Proceedings. Seventh Pacific Conference on Computer Graphics and Applications (Cat. No.PR00293), 1999
Volume visualization is an essential part of many computer-assisted medical applications. While g... more Volume visualization is an essential part of many computer-assisted medical applications. While generalpurpose commercial visualization software can provide common functions for visualizing three-dimensional (3D) medical data, there are many occasions where speci c requirements cannot be ful lled by such s o f t ware. In orthopaedic surgery involving screw insertion, surgeons must consider not only the surface topology but also the bone mineral density (BMD) distribution to ensure a high success rate of the operation. In this paper we p r e s e n t a new system, called VISBONE, for visualizing the 3D BMD distribution within a bone. Design issues and speci c considerations required by this application are discussed.
Borrelia burgdorferi, the agent of Lyme disease, spreads from the site of the tick bite to tissue... more Borrelia burgdorferi, the agent of Lyme disease, spreads from the site of the tick bite to tissues such as heart, joints and the nervous system. Host glycosaminoglycans (GAGs), highly modified repeating disaccharides that are present on cell surfaces and in extracellular matrix, are common targets of microbial pathogens during tissue colonization. While several dermatan sulfate-binding B. burgdorferi adhesins have been identified, B. burgdorferi adhesins documented to promote spirochetal binding to heparan sulfate have not yet been identified. OspEF-related proteins (Erps), a large family of plasmid-encoded surface lipoproteins that are produced in the mammalian host, can be divided into the OspF-related, OspEF leader peptide (Elp), and OspE-related subfamilies. We show here that a member of the OspF-related subfamily, ErpG, binds to heparan sulfate, and when produced on the surface of an otherwise nonadherent B. burgdorferi strain, ErpG promotes heparan sulfate-mediated bacterial a...
The ability of the Lyme disease spirochete to attach to host components may contribute to its abi... more The ability of the Lyme disease spirochete to attach to host components may contribute to its ability to infect diverse tissues. We present evidence that the Lyme disease spirochete expresses a lectin activity that promotes agglutination of erythrocytes and bacterial attachment to glycosaminoglycans. Among a diverse collection of 21 strains of Lyme disease spirochete, hemagglutinating activity was easily detected in all but 3 strains, and these three strains were noninfectious. The ability to agglutinate erythrocytes was associated with the ability of the spirochete to bind to the sulfated polysaccharide dextran sulfate and to mammalian cells. Soluble dextran sulfate was a potent inhibitor of both hemagglutination and attachment to mammalian cells, while dextran had no effect on either activity, suggesting that dextran sulfate may inhibit attachment by mimicking host cell glycosaminoglycans. Consistent with this, the spirochete bound to immobilized heparin, and soluble heparin inhib...
Decorin-binding protein A (DbpA) of Borrelia burgdorferi mediates bacterial adhesion to heparin a... more Decorin-binding protein A (DbpA) of Borrelia burgdorferi mediates bacterial adhesion to heparin and dermatan sulfate associated with decorin. Lysines K82, K163, and K170 of DbpA are known to be important for in vitro interaction with decorin, and the DbpA structure, initially solved by nuclear magnetic resonance (NMR) spectroscopy, suggests these lysine residues colocalize in a pocket near the C terminus of the protein. In the current study, we solved the structure of DbpA from B. burgdorferi strain 297 using X-ray crystallography and confirmed the existing NMR structural data. In vitro binding experiments confirmed that recombinant DbpA proteins with mutations in K82, K163, or K170 did not bind decorin, which was due to an inability to interact with dermatan sulfate. Most importantly, we determined that the in vitro binding defect observed upon mutation of K82, K163, or K170 in DbpA also led to a defect during infection. The infectivity of B. burgdorferi expressing individual dbpA ...
The Lyme disease spirochete, Borrelia burgdorferi, infects multiple tissues, such as the heart, j... more The Lyme disease spirochete, Borrelia burgdorferi, infects multiple tissues, such as the heart, joint, skin, and nervous system and has been shown to recognize heparan sulfate and dermatan sulfate proteoglycans. In this study, we examined the contribution of different classes of proteoglycans to the attachment of the infectious B. burgdorferi strain N40 to several immortalized cell lines and primary cultured cells, including endothelial cells and brain cells. Bacterial attachment was inhibited by exogenous proteoglycans or by treatment of host cells with inhibitors of proteoglycan synthesis or sulfation, indicating that proteoglycans play a critical role in bacterial binding to diverse cell types. Binding to primary bovine capillary endothelial cells or a human endothelial cell line was also inhibited by digestion with heparinase or heparitinase but not with chondroitinase ABC. In contrast, binding to glial cell-enriched brain cell cultures or to a neuronal cell line was inhibited b...
The immunoglobulin G (IgG) antibody response to outer surface protein A (OspA) of Borrelia burgdo... more The immunoglobulin G (IgG) antibody response to outer surface protein A (OspA) of Borrelia burgdorferi has been reported to occur late in the course of Lyme disease. To learn when reactivity to particular epitopes of OspA develops and whether the strength of particular responses correlates with the duration of arthritis and HLA-DR specificities, we determined the IgM and IgG responses by enzyme-linked immunosorbent assay in 128 patients with various manifestations of Lyme disease to full-length recombinant OspA and three OspA fragments which divided the protein approximately into thirds. Among the 10 patients who were followed serially, an early IgM response was often found to epitopes in all three fragments of OspA, sometimes accompanied by a weak IgG response, primarily to an epitope in the middle third of the protein. Months to years later, the seven patients who had prolonged or moderate episodes of arthritis developed strong IgG responses to OspA, especially to epitopes in the ...
Chronic Lyme arthritis that is unresponsive to antibiotic therapy is associated with an increased... more Chronic Lyme arthritis that is unresponsive to antibiotic therapy is associated with an increased frequency of the HLA-DR4 specificity. To determine whether the immune response to a particular polypeptide of Borrelia burgdorferi may be associated with treatment-resistant chronic Lyme arthritis, we correlated the clinical courses and HLA-DR specificities of 128 patients with Lyme disease with their antibody responses to spirochetal polypeptides. Antibody reactivity was determined by Western blotting (immunoblotting) with sonicated whole B. burgdorferi and recombinant forms of its outer surface proteins, OspA and OspB, as the antigen preparations. Of 15 patients monitored for 4 to 12 years, 11 (73%) developed strong immunoglobulin G responses to both OspA and OspB near the beginning of prolonged episodes of arthritis, from 5 months to 7 years after disease onset. When single serum samples from 80 patients with Lyme arthritis, were tested, 57 (71%) showed antibody reactivity to recombi...
Microbial pathogens that colonize multiple tissues commonly produce adhesive surface proteins tha... more Microbial pathogens that colonize multiple tissues commonly produce adhesive surface proteins that mediate attachment to cells and/or extracellular matrix in target organs. Many of these 'adhesins' bind to multiple ligands, complicating efforts to understand the role of each ligand-binding activity. Borrelia burgdorferi, the causative agent of Lyme disease, produces BBK32, first identified as a fibronectin-binding adhesin that promotes skin and joint colonization. BBK32 also binds to glycosaminoglycan (GAG), which, like fibronectin is ubiquitously present on cell surfaces. To determine which binding activity is relevant for BBK32-promoted infectivity, we generated a panel of BBK32 truncation and internal deletion mutants, and identified variants specifically defective for binding to either fibronectin or GAG. These variants promoted bacterial attachment to different mammalian cell types in vitro, suggesting that fibronectin and GAG binding may play distinct roles during infe...
Shiga toxins (Stx) are a family of cytotoxic proteins that can cause Hemolytic Uremic Syndrome 13... more Shiga toxins (Stx) are a family of cytotoxic proteins that can cause Hemolytic Uremic Syndrome 13 (HUS), a thrombotic microangiopathy, following infections by Shiga toxin producing 14 Escherichia coli (STEC). Renal failure is a key feature of HUS and a major cause of childhood 15 renal failure worldwide. There are currently no specific therapies for STEC-associated HUS and 16 the mechanism of Stx-induced renal injury is not well understood primarily due to a lack of fully 17 representative animal models and an inability to monitor disease progression on a molecular or 18 cellular level in humans at early stages. 3D tissue models have been shown to be more in vivo-19 like in their phenotype and physiology than 2D cultures for numerous disease models including 20 cancer and polycystic kidney disease. It is unknown if exposure of a 3D renal tissue model to Stx 21 will yield a more in vivo-like response than 2D cell culture. In this study, we have characterized 22 Stx2-mediated cytotoxicity in a bioengineered 3D human renal tissue model previously shown to 23 be a predictor of drug-induced nephrotoxicity and compared its response to Stx2 exposure in 2D 24 cell culture. Our results demonstrate that although many mechanistic aspects of cytotoxicity were 25 similar between 3D and 2D, treatment of the 3D tissues with Stx resulted in an elevated secretion 26 of the kidney injury marker 1 (Kim-1) and the cytokine IL-8 compared to the 2D cell cultures.
Lyme disease spirochetes demonstrate strain-and species-specific differences in tissue tropism. F... more Lyme disease spirochetes demonstrate strain-and species-specific differences in tissue tropism. For example, the three major Lyme disease spirochete species, Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii, are each most commonly associated with overlapping but distinct spectra of clinical manifestations. Borrelia burgdorferi sensu stricto, the most common Lyme spirochete in the U.S., is closely associated with arthritis.
Enterohemorrhagic Escherichia coli (EHEC) generate F-actin-rich adhesion pedestals by delivering ... more Enterohemorrhagic Escherichia coli (EHEC) generate F-actin-rich adhesion pedestals by delivering effector proteins into mammalian cells. These effectors include the translocated receptor Tir, along with EspF U , a protein that associates indirectly with Tir and contains multiple peptide repeats that stimulate actin polymerization. In vitro, the EspF U repeat region is capable of binding and activating recombinant derivatives of N-WASP, a host actin nucleation-promoting factor. In spite of the identification of these important bacterial and host factors, the underlying mechanisms of how EHEC so potently exploits the native actin assembly machinery have not been clearly defined. Here we show that Tir and EspF U are sufficient for actin pedestal formation in cultured cells. Experimental clustering of Tir-EspF U fusion proteins indicates that the central role of the cytoplasmic portion of Tir is to promote clustering of the repeat region of EspF U . Whereas clustering of a single EspF U repeat is sufficient to bind N-WASP and generate pedestals on cultured cells, multi-repeat EspF U derivatives promote actin assembly more efficiently. Moreover, the EspF U repeats activate a protein complex containing N-WASP and the actinbinding protein WIP in a synergistic fashion in vitro, further suggesting that the repeats cooperate to stimulate actin polymerization in vivo. One explanation for repeat synergy is that simultaneous engagement of multiple N-WASP molecules can enhance its ability to interact with the actin nucleating Arp2/3 complex. These findings define the minimal set of bacterial effectors required for pedestal formation and the elements within those effectors that contribute to actin assembly via N-WASP-Arp2/3-mediated signaling pathways.
Background: Non-muscle myosin IIA is involved in force generation, movement, and membrane reshapi... more Background: Non-muscle myosin IIA is involved in force generation, movement, and membrane reshaping. Its activity is regulated by phosphorylation of the light chain. Results: NMHC-IIA head domain is tyrosine-phosphorylated by Src and modulates Listeria intracellular levels. Conclusion: Tyrosine phosphorylation of NMHC-IIA affects the outcome of infection. Significance: This novel post-translational modification of NMHC-IIA possibly affects its functions. Downloaded from 6 The abbreviations used are: NMHC-IIA, non-muscle myosin heavy chain IIA; NMHC-IIB, non-muscle myosin heavy chain IIB; WCL, whole cell lysate; m.o.i.
Journal of immunology (Baltimore, Md. : 1950), 2015
Streptococcus pneumoniae infections are an important cause of morbidity and mortality in older pa... more Streptococcus pneumoniae infections are an important cause of morbidity and mortality in older patients. Uncontrolled neutrophil-driven pulmonary inflammation exacerbates this disease. To test whether the α-tocopherol (α-Toc) form of vitamin E, a regulator of immunity, can modulate neutrophil responses as a preventive strategy to mitigate the age-associated decline in resistance to S. pneumoniae, young (4 mo) and old (22-24 mo) C57BL/6 mice were fed a diet containing 30-PPM (control) or 500-PPM (supplemented) α-Toc for 4 wk and intratracheally infected with S. pneumoniae. Aged mice fed a control diet were exquisitely more susceptible to S. pneumoniae than young mice. At 2 d postinfection, aged mice suffered 1000-fold higher pulmonary bacterial burden, 2.2-fold higher levels of neutrophil recruitment to the lung, and a 2.25-fold higher rate of lethal septicemia. Strikingly, α-Toc supplementation of aged mice resulted in a 1000-fold lower bacterial lung burden and full control of infe...
Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have b... more Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.
An important determinant of disease following Streptococcus pneumoniae (pneumococcus) lung infect... more An important determinant of disease following Streptococcus pneumoniae (pneumococcus) lung infection is pulmonary inflammation mediated by polymorphonuclear leukocytes (PMNs). We found that upon intratracheal challenge of mice, recruitment of PMNs into the lungs within the first 3 hours coincided with decreased pulmonary pneumococci, whereas large numbers of pulmonary PMNs beyond 12 hours correlated with a greater bacterial burden. Indeed, mice that survived infection largely resolved inflammation by 72 hours, and PMN depletion at peak infiltration, i.e. 18 hours post-infection, lowered bacterial numbers and enhanced survival. We investigated host signaling pathways that influence both pneumococcus clearance and pulmonary inflammation. Pharmacologic inhibition and/or genetic ablation of enzymes that generate extracellular adenosine (EAD) (e.g. the ectoenzyme CD73) or degrade EAD (e.g. adenosine deaminase) revealed that EAD dramatically increases murine resistance to S. pneumoniae lu...
Medical science monitor: international medical journal of experimental and clinical research
Gait analysis techniques guide the use and design of functional electrical stimulation (FES) syst... more Gait analysis techniques guide the use and design of functional electrical stimulation (FES) systems for paraplegic walking. However, published studies on dynamic gait stability for the effective use of FES are limited. This paper introduces a new risk-tendency graph (RTG) technique to analyze and process gait stability in FES-assisted paraplegic walking. The main instrument was a specialized walker dynamometer system based on a multi-channel strain-gauge bridge network affixed to the frame of the walker. This system collects force information for the handle reaction vector (HRV) between the patient's upper extremities and the walker during walking. The information is then converted into a walker tipping index (WTI), which is an indicator of the patient's walking stability. Dynamic gait stability is then combined with spatio-temporal locating methods for WTI and visually described as morphological curves in the temporal and spatial domains, namely RTGs. To demonstrate the po...
This study evaluated the biomechanical and electromyographic effects of conventional ankle foot o... more This study evaluated the biomechanical and electromyographic effects of conventional ankle foot orthoses (AFOs) and dynamic ankle foot orthoses (DAFOs) on gait in patients with spastic cerebral palsy (CP). Thirteen patients with dynamic equinus underwent motion analysis with electromyography. Both AFOs and DAFOs provided longer stride length, permitted pre-positioning for initial contact, and successfully controlled the excessive plantarflexion during the swing phase. Median frequency (MF) of EMG signal indicated that extremely high firing was found in the patient's lower limbs compared to controls that resulted in tiredness. The DAFOs allowed a significantly larger total ankle range of motion than the AFOs. However, AFOs significantly reduced the MF while DAFOs did not. The reduced MF seen when wearing AFOs suggested an improvement of walking endurance. The DAFO had the advantage of less restriction on ankle movement, which avoids muscular atrophy and improves orthotic compliance. #
Borrelia burgdorferi surface-located membrane protein 1, also known as Lmp1, has been shown to pl... more Borrelia burgdorferi surface-located membrane protein 1, also known as Lmp1, has been shown to play critical roles in pathogen evasion of host-acquired immune defences, thereby facilitating persistent infection. Lmp1 possesses three regions representing potentially discrete domains: Lmp1N, Lmp1M and Lmp1C. Because of its insignificant homology to known proteins, how Lmp1 or its specific regions contribute to microbial biology and infection remains enigmatic. Here, we show that distinct from Lmp1N and Lmp1C, Lmp1M is composed of at least 70% alpha helices and completely lacks recognizable beta sheets. The region binds to host glycosaminoglycan chondroitin-6-sulfate molecules and facilitates mammalian cell attachment, suggesting an adhesin function of Lmp1M. Phenotypic analysis of the Lmp1-deficient mutant engineered to produce Lmp1M on the microbial surface suggests that Lmp1M can independently support B. burgdorferi infectivity in murine hosts. Further exploration of functions of Lmp1 distinct regions will shed new light on the intriguing biology and infectivity of spirochetes and help develop novel interventions to combat Lyme disease.
Proceedings. Seventh Pacific Conference on Computer Graphics and Applications (Cat. No.PR00293), 1999
Volume visualization is an essential part of many computer-assisted medical applications. While g... more Volume visualization is an essential part of many computer-assisted medical applications. While generalpurpose commercial visualization software can provide common functions for visualizing three-dimensional (3D) medical data, there are many occasions where speci c requirements cannot be ful lled by such s o f t ware. In orthopaedic surgery involving screw insertion, surgeons must consider not only the surface topology but also the bone mineral density (BMD) distribution to ensure a high success rate of the operation. In this paper we p r e s e n t a new system, called VISBONE, for visualizing the 3D BMD distribution within a bone. Design issues and speci c considerations required by this application are discussed.
Borrelia burgdorferi, the agent of Lyme disease, spreads from the site of the tick bite to tissue... more Borrelia burgdorferi, the agent of Lyme disease, spreads from the site of the tick bite to tissues such as heart, joints and the nervous system. Host glycosaminoglycans (GAGs), highly modified repeating disaccharides that are present on cell surfaces and in extracellular matrix, are common targets of microbial pathogens during tissue colonization. While several dermatan sulfate-binding B. burgdorferi adhesins have been identified, B. burgdorferi adhesins documented to promote spirochetal binding to heparan sulfate have not yet been identified. OspEF-related proteins (Erps), a large family of plasmid-encoded surface lipoproteins that are produced in the mammalian host, can be divided into the OspF-related, OspEF leader peptide (Elp), and OspE-related subfamilies. We show here that a member of the OspF-related subfamily, ErpG, binds to heparan sulfate, and when produced on the surface of an otherwise nonadherent B. burgdorferi strain, ErpG promotes heparan sulfate-mediated bacterial a...
The ability of the Lyme disease spirochete to attach to host components may contribute to its abi... more The ability of the Lyme disease spirochete to attach to host components may contribute to its ability to infect diverse tissues. We present evidence that the Lyme disease spirochete expresses a lectin activity that promotes agglutination of erythrocytes and bacterial attachment to glycosaminoglycans. Among a diverse collection of 21 strains of Lyme disease spirochete, hemagglutinating activity was easily detected in all but 3 strains, and these three strains were noninfectious. The ability to agglutinate erythrocytes was associated with the ability of the spirochete to bind to the sulfated polysaccharide dextran sulfate and to mammalian cells. Soluble dextran sulfate was a potent inhibitor of both hemagglutination and attachment to mammalian cells, while dextran had no effect on either activity, suggesting that dextran sulfate may inhibit attachment by mimicking host cell glycosaminoglycans. Consistent with this, the spirochete bound to immobilized heparin, and soluble heparin inhib...
Decorin-binding protein A (DbpA) of Borrelia burgdorferi mediates bacterial adhesion to heparin a... more Decorin-binding protein A (DbpA) of Borrelia burgdorferi mediates bacterial adhesion to heparin and dermatan sulfate associated with decorin. Lysines K82, K163, and K170 of DbpA are known to be important for in vitro interaction with decorin, and the DbpA structure, initially solved by nuclear magnetic resonance (NMR) spectroscopy, suggests these lysine residues colocalize in a pocket near the C terminus of the protein. In the current study, we solved the structure of DbpA from B. burgdorferi strain 297 using X-ray crystallography and confirmed the existing NMR structural data. In vitro binding experiments confirmed that recombinant DbpA proteins with mutations in K82, K163, or K170 did not bind decorin, which was due to an inability to interact with dermatan sulfate. Most importantly, we determined that the in vitro binding defect observed upon mutation of K82, K163, or K170 in DbpA also led to a defect during infection. The infectivity of B. burgdorferi expressing individual dbpA ...
The Lyme disease spirochete, Borrelia burgdorferi, infects multiple tissues, such as the heart, j... more The Lyme disease spirochete, Borrelia burgdorferi, infects multiple tissues, such as the heart, joint, skin, and nervous system and has been shown to recognize heparan sulfate and dermatan sulfate proteoglycans. In this study, we examined the contribution of different classes of proteoglycans to the attachment of the infectious B. burgdorferi strain N40 to several immortalized cell lines and primary cultured cells, including endothelial cells and brain cells. Bacterial attachment was inhibited by exogenous proteoglycans or by treatment of host cells with inhibitors of proteoglycan synthesis or sulfation, indicating that proteoglycans play a critical role in bacterial binding to diverse cell types. Binding to primary bovine capillary endothelial cells or a human endothelial cell line was also inhibited by digestion with heparinase or heparitinase but not with chondroitinase ABC. In contrast, binding to glial cell-enriched brain cell cultures or to a neuronal cell line was inhibited b...
The immunoglobulin G (IgG) antibody response to outer surface protein A (OspA) of Borrelia burgdo... more The immunoglobulin G (IgG) antibody response to outer surface protein A (OspA) of Borrelia burgdorferi has been reported to occur late in the course of Lyme disease. To learn when reactivity to particular epitopes of OspA develops and whether the strength of particular responses correlates with the duration of arthritis and HLA-DR specificities, we determined the IgM and IgG responses by enzyme-linked immunosorbent assay in 128 patients with various manifestations of Lyme disease to full-length recombinant OspA and three OspA fragments which divided the protein approximately into thirds. Among the 10 patients who were followed serially, an early IgM response was often found to epitopes in all three fragments of OspA, sometimes accompanied by a weak IgG response, primarily to an epitope in the middle third of the protein. Months to years later, the seven patients who had prolonged or moderate episodes of arthritis developed strong IgG responses to OspA, especially to epitopes in the ...
Chronic Lyme arthritis that is unresponsive to antibiotic therapy is associated with an increased... more Chronic Lyme arthritis that is unresponsive to antibiotic therapy is associated with an increased frequency of the HLA-DR4 specificity. To determine whether the immune response to a particular polypeptide of Borrelia burgdorferi may be associated with treatment-resistant chronic Lyme arthritis, we correlated the clinical courses and HLA-DR specificities of 128 patients with Lyme disease with their antibody responses to spirochetal polypeptides. Antibody reactivity was determined by Western blotting (immunoblotting) with sonicated whole B. burgdorferi and recombinant forms of its outer surface proteins, OspA and OspB, as the antigen preparations. Of 15 patients monitored for 4 to 12 years, 11 (73%) developed strong immunoglobulin G responses to both OspA and OspB near the beginning of prolonged episodes of arthritis, from 5 months to 7 years after disease onset. When single serum samples from 80 patients with Lyme arthritis, were tested, 57 (71%) showed antibody reactivity to recombi...
Microbial pathogens that colonize multiple tissues commonly produce adhesive surface proteins tha... more Microbial pathogens that colonize multiple tissues commonly produce adhesive surface proteins that mediate attachment to cells and/or extracellular matrix in target organs. Many of these 'adhesins' bind to multiple ligands, complicating efforts to understand the role of each ligand-binding activity. Borrelia burgdorferi, the causative agent of Lyme disease, produces BBK32, first identified as a fibronectin-binding adhesin that promotes skin and joint colonization. BBK32 also binds to glycosaminoglycan (GAG), which, like fibronectin is ubiquitously present on cell surfaces. To determine which binding activity is relevant for BBK32-promoted infectivity, we generated a panel of BBK32 truncation and internal deletion mutants, and identified variants specifically defective for binding to either fibronectin or GAG. These variants promoted bacterial attachment to different mammalian cell types in vitro, suggesting that fibronectin and GAG binding may play distinct roles during infe...
Shiga toxins (Stx) are a family of cytotoxic proteins that can cause Hemolytic Uremic Syndrome 13... more Shiga toxins (Stx) are a family of cytotoxic proteins that can cause Hemolytic Uremic Syndrome 13 (HUS), a thrombotic microangiopathy, following infections by Shiga toxin producing 14 Escherichia coli (STEC). Renal failure is a key feature of HUS and a major cause of childhood 15 renal failure worldwide. There are currently no specific therapies for STEC-associated HUS and 16 the mechanism of Stx-induced renal injury is not well understood primarily due to a lack of fully 17 representative animal models and an inability to monitor disease progression on a molecular or 18 cellular level in humans at early stages. 3D tissue models have been shown to be more in vivo-19 like in their phenotype and physiology than 2D cultures for numerous disease models including 20 cancer and polycystic kidney disease. It is unknown if exposure of a 3D renal tissue model to Stx 21 will yield a more in vivo-like response than 2D cell culture. In this study, we have characterized 22 Stx2-mediated cytotoxicity in a bioengineered 3D human renal tissue model previously shown to 23 be a predictor of drug-induced nephrotoxicity and compared its response to Stx2 exposure in 2D 24 cell culture. Our results demonstrate that although many mechanistic aspects of cytotoxicity were 25 similar between 3D and 2D, treatment of the 3D tissues with Stx resulted in an elevated secretion 26 of the kidney injury marker 1 (Kim-1) and the cytokine IL-8 compared to the 2D cell cultures.
Lyme disease spirochetes demonstrate strain-and species-specific differences in tissue tropism. F... more Lyme disease spirochetes demonstrate strain-and species-specific differences in tissue tropism. For example, the three major Lyme disease spirochete species, Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii, are each most commonly associated with overlapping but distinct spectra of clinical manifestations. Borrelia burgdorferi sensu stricto, the most common Lyme spirochete in the U.S., is closely associated with arthritis.
Enterohemorrhagic Escherichia coli (EHEC) generate F-actin-rich adhesion pedestals by delivering ... more Enterohemorrhagic Escherichia coli (EHEC) generate F-actin-rich adhesion pedestals by delivering effector proteins into mammalian cells. These effectors include the translocated receptor Tir, along with EspF U , a protein that associates indirectly with Tir and contains multiple peptide repeats that stimulate actin polymerization. In vitro, the EspF U repeat region is capable of binding and activating recombinant derivatives of N-WASP, a host actin nucleation-promoting factor. In spite of the identification of these important bacterial and host factors, the underlying mechanisms of how EHEC so potently exploits the native actin assembly machinery have not been clearly defined. Here we show that Tir and EspF U are sufficient for actin pedestal formation in cultured cells. Experimental clustering of Tir-EspF U fusion proteins indicates that the central role of the cytoplasmic portion of Tir is to promote clustering of the repeat region of EspF U . Whereas clustering of a single EspF U repeat is sufficient to bind N-WASP and generate pedestals on cultured cells, multi-repeat EspF U derivatives promote actin assembly more efficiently. Moreover, the EspF U repeats activate a protein complex containing N-WASP and the actinbinding protein WIP in a synergistic fashion in vitro, further suggesting that the repeats cooperate to stimulate actin polymerization in vivo. One explanation for repeat synergy is that simultaneous engagement of multiple N-WASP molecules can enhance its ability to interact with the actin nucleating Arp2/3 complex. These findings define the minimal set of bacterial effectors required for pedestal formation and the elements within those effectors that contribute to actin assembly via N-WASP-Arp2/3-mediated signaling pathways.
Background: Non-muscle myosin IIA is involved in force generation, movement, and membrane reshapi... more Background: Non-muscle myosin IIA is involved in force generation, movement, and membrane reshaping. Its activity is regulated by phosphorylation of the light chain. Results: NMHC-IIA head domain is tyrosine-phosphorylated by Src and modulates Listeria intracellular levels. Conclusion: Tyrosine phosphorylation of NMHC-IIA affects the outcome of infection. Significance: This novel post-translational modification of NMHC-IIA possibly affects its functions. Downloaded from 6 The abbreviations used are: NMHC-IIA, non-muscle myosin heavy chain IIA; NMHC-IIB, non-muscle myosin heavy chain IIB; WCL, whole cell lysate; m.o.i.
Journal of immunology (Baltimore, Md. : 1950), 2015
Streptococcus pneumoniae infections are an important cause of morbidity and mortality in older pa... more Streptococcus pneumoniae infections are an important cause of morbidity and mortality in older patients. Uncontrolled neutrophil-driven pulmonary inflammation exacerbates this disease. To test whether the α-tocopherol (α-Toc) form of vitamin E, a regulator of immunity, can modulate neutrophil responses as a preventive strategy to mitigate the age-associated decline in resistance to S. pneumoniae, young (4 mo) and old (22-24 mo) C57BL/6 mice were fed a diet containing 30-PPM (control) or 500-PPM (supplemented) α-Toc for 4 wk and intratracheally infected with S. pneumoniae. Aged mice fed a control diet were exquisitely more susceptible to S. pneumoniae than young mice. At 2 d postinfection, aged mice suffered 1000-fold higher pulmonary bacterial burden, 2.2-fold higher levels of neutrophil recruitment to the lung, and a 2.25-fold higher rate of lethal septicemia. Strikingly, α-Toc supplementation of aged mice resulted in a 1000-fold lower bacterial lung burden and full control of infe...
Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have b... more Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.
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Papers by John Leong