Publications by authors named "John M Leong"

94 Publications

Citrobacter rodentium Lysogenized with a Shiga Toxin-Producing Phage: A Murine Model for Shiga Toxin-Producing E. coli Infection.

Methods Mol Biol 2021 ;2291:381-397

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USA.

Shiga toxin-producing E. coli (STEC) is a common foodborne pathogen in developed countries. STEC generates "attaching and effacing" (AE) lesions on colonic epithelium, characterized by effacement of microvilli and the formation of actin "pedestals" beneath intimately attached bacteria. In addition, STEC are lysogenized with a phage that, upon induction, can produce potent Shiga toxins (Stx), potentially leading to both hemorrhagic colitis and hemolytic uremic syndrome. Investigation of the pathogenesis of this disease has been challenging because STEC does not readily colonize conventional mice.Citrobacter rodentium (CR) is a related mouse pathogen that also generates AE lesions. Whereas CR does not produce Stx, a murine model for STEC utilizes CR lysogenized with an E. coli-derived Stx phage, generating CR(Φstx), which both colonizes conventional mice and readily gives rise to systemic disease. We present here key methods for the use of CR(Φstx) infection as a highly predictable murine model for infection and disease by STEC. Importantly, we detail CR(Φstx) inoculation by feeding, determination of pathogen colonization, production of phage and toxin, and assessment of intestinal and renal pathology. These methods provide a framework for studying STEC-mediated systemic disease that may aid in the development of efficacious therapeutics.
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http://dx.doi.org/10.1007/978-1-0716-1339-9_19DOI Listing
April 2021

Safe and effective delivery of supplemental iron to healthy older adults: The double-blind, randomized, placebo-controlled trial protocol of the Safe Iron Study.

Gates Open Res 2019 9;3:1510. Epub 2021 Feb 9.

Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, 02111, USA.

The forms of iron currently available to correct iron deficiency have adverse effects, including infectious diarrhea, increased susceptibility to malaria, inflammation and detrimental changes to the gut microbiome. These adverse effects limit their use such that the growing burden of iron deficiency has not abated in recent decades. Here, we summarize the protocol of the "Safe Iron Study", the first clinical study examining the safety and efficacy of novel forms of iron in healthy, iron-replete adults. The Safe Iron Study is a double-blind, randomized, placebo-controlled trial conducted in Boston, MA, USA. This study compares ferrous sulfate heptahydrate (FeSO ·H O) with two novel forms of iron supplements (iron hydroxide adipate tartrate (IHAT) and organic fungal iron metabolite (Aspiron™ Natural Koji Iron)). In Phase I, we will compare each source of iron administrated at a low dose (60 mg Fe/day). We will also determine the effect of FeSO co-administrated with a multiple micronutrient powder and weekly administration of FeSO . The forms of iron found to produce no adverse effects, or adverse effects no greater than FeSO in Phase I, Phase II will evaluate a higher, i.e., a therapeutic dose (120 mg Fe/day). The primary outcomes of this study include malaria ( ) infectivity of host erythrocytes, bacterial proliferation (of selected species) in presence of host plasma and intestinal inflammation assessed by fecal calprotectin. This study will test the hypotheses that the novel forms of iron, administered at equivalent doses to FeSO , will produce similar increases in iron status in iron-replete subjects, yet lower increases in malaria infectivity, bacterial proliferation, gut inflammation. Ultimately, this study seeks to contribute to development of safe and effective forms of supplemental iron to address the global burden of iron deficiency and anemia. ClinicalTrials.gov identifier: NCT03212677; registered: 11 July 2017.
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http://dx.doi.org/10.12688/gatesopenres.13039.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890045PMC
February 2021

Macrophage LC3-associated phagocytosis is an immune defense against that diminishes with host aging.

Proc Natl Acad Sci U S A 2020 12 21;117(52):33561-33569. Epub 2020 Dec 21.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111;

is a leading cause of pneumonia and invasive disease, particularly, in the elderly. lung infection of aged mice is associated with high bacterial burdens and detrimental inflammatory responses. Macrophages can clear microorganisms and modulate inflammation through two distinct lysosomal trafficking pathways that involve 1A/1B-light chain 3 (LC3)-marked organelles, canonical autophagy, and LC3-associated phagocytosis (LAP). The pore-forming toxin pneumolysin (PLY) triggers an autophagic response in nonphagocytic cells, but the role of LAP in macrophage defense against or in age-related susceptibility to infection is unexplored. We found that infection of murine bone-marrow-derived macrophages (BMDMs) by PLY-producing triggered Atg5- and Atg7-dependent recruitment of LC3 to -containing vesicles. The association of LC3 with -containing phagosomes required components specific for LAP, such as Rubicon and the NADPH oxidase, but not factors, such as Ulk1, FIP200, or Atg14, required specifically for canonical autophagy. In addition, was sequestered within single-membrane compartments indicative of LAP. Importantly, compared to BMDMs from young (2-mo-old) mice, BMDMs from aged (20- to 22-mo-old) mice infected with were not only deficient in LAP and bacterial killing, but also produced higher levels of proinflammatory cytokines. Inhibition of LAP enhanced survival and cytokine responses in BMDMs from young but not aged mice. Thus, LAP is an important innate immune defense employed by BMDMs to control infection and concomitant inflammation, one that diminishes with age and may contribute to age-related susceptibility to this important pathogen.
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http://dx.doi.org/10.1073/pnas.2015368117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7776987PMC
December 2020

Strain-specific joint invasion and colonization by Lyme disease spirochetes is promoted by outer surface protein C.

PLoS Pathog 2020 05 15;16(5):e1008516. Epub 2020 May 15.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

Lyme disease, caused by Borrelia burgdorferi, B. afzelii and B. garinii, is a chronic, multi-systemic infection and the spectrum of tissues affected can vary with the Lyme disease strain. For example, whereas B. garinii infection is associated with neurologic manifestations, B. burgdorferi infection is associated with arthritis. The basis for tissue tropism is poorly understood, but has been long hypothesized to involve strain-specific interactions with host components in the target tissue. OspC (outer surface protein C) is a highly variable outer surface protein required for infectivity, and sequence differences in OspC are associated with variation in tissue invasiveness, but whether OspC directly influences tropism is unknown. We found that OspC binds to the extracellular matrix (ECM) components fibronectin and/or dermatan sulfate in an OspC variant-dependent manner. Murine infection by isogenic B. burgdorferi strains differing only in their ospC coding region revealed that two OspC variants capable of binding dermatan sulfate promoted colonization of all tissues tested, including joints. However, an isogenic strain producing OspC from B. garinii strain PBr, which binds fibronectin but not dermatan sulfate, colonized the skin, heart and bladder, but not joints. Moreover, a strain producing an OspC altered to recognize neither fibronectin nor dermatan sulfate displayed dramatically reduced levels of tissue colonization that were indistinguishable from a strain entirely deficient in OspC. Finally, intravital microscopy revealed that this OspC mutant, in contrast to a strain producing wild type OspC, was defective in promoting joint invasion by B. burgdorferi in living mice. We conclude that OspC functions as an ECM-binding adhesin that is required for joint invasion, and that variation in OspC sequence contributes to strain-specific differences in tissue tropism displayed among Lyme disease spirochetes.
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http://dx.doi.org/10.1371/journal.ppat.1008516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255614PMC
May 2020

Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production.

J Leukoc Biol 2020 09 4;108(3):867-882. Epub 2020 Feb 4.

Department of Microbiology and Immunology, University at Buffalo School of Medicine, Buffalo, New York, USA.

Polymorphonuclear leukocytes (PMNs) are crucial for initial control of Streptococcus pneumoniae (pneumococcus) lung infection; however, as the infection progresses their persistence in the lungs becomes detrimental. Here we explored why the antimicrobial efficacy of PMNs declines over the course of infection. We found that the progressive inability of PMNs to control infection correlated with phenotypic differences characterized by a decrease in CD73 expression, an enzyme required for production of extracellular adenosine (EAD). EAD production by CD73 was crucial for the ability of both murine and human PMNs to kill S. pneumoniae. In exploring the mechanisms by which CD73 controlled PMN function, we found that CD73 mediated its antimicrobial activity by inhibiting IL-10 production. PMNs from wild-type mice did not increase IL-10 production in response to S. pneumoniae; however, CD73 PMNs up-regulated IL-10 production upon pneumococcal infection in vitro and during lung challenge. IL-10 inhibited the ability of WT PMNs to kill pneumococci. Conversely, blocking IL-10 boosted the bactericidal activity of CD73 PMNs as well as host resistance of CD73 mice to pneumococcal pneumonia. CD73/IL-10 did not affect apoptosis, bacterial uptake, and intracellular killing or production of antimicrobial neutrophil elastase and myeloperoxidase. Rather, inhibition of IL-10 production by CD73 was important for optimal reactive oxygen species (ROS) production by PMNs. ROS contributed to PMN antimicrobial function as their removal or detoxification impaired the ability of PMNs to efficiently kill S. pneumoniae. This study demonstrates that CD73 controls PMN antimicrobial phenotype during S. pneumoniae infection.
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http://dx.doi.org/10.1002/JLB.4MA0120-115RRDOI Listing
September 2020

Pneumolysin Induces 12-Lipoxygenase-Dependent Neutrophil Migration during Infection.

J Immunol 2020 01 27;204(1):101-111. Epub 2019 Nov 27.

Department of Molecular Biology and Microbiology, Tufts University, Boston, MA 02111;

is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)-dependent lipid inflammatory mediator hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major virulence factor that generates ∼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient mutant was impaired in triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the α-toxin of , which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non-pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX-dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOX-dependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX-deficient mice indicates that 12-LOX-independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indicate that PLY is an important factor in promoting hepoxilin A3-dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion.
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http://dx.doi.org/10.4049/jimmunol.1800748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195902PMC
January 2020

A1 adenosine receptor signaling reduces Streptococcus pneumoniae adherence to pulmonary epithelial cells by targeting expression of platelet-activating factor receptor.

Cell Microbiol 2020 02 20;22(2):e13141. Epub 2019 Nov 20.

Department of Microbiology and Immunology, State University of New York at Buffalo School of Medicine, Buffalo, New York.

Extracellular adenosine production is crucial for host resistance against Streptococcus pneumoniae (pneumococcus) and is thought to affect antibacterial immune responses by neutrophils. However, whether extracellular adenosine alters direct host-pathogen interaction remains unexplored. An important determinant for lung infection by S. pneumoniae is its ability to adhere to the pulmonary epithelium. Here we explored whether extracellular adenosine can directly impact bacterial adherence to lung epithelial cells. We found that signaling via A1 adenosine receptor significantly reduced the ability of pneumococci to bind human pulmonary epithelial cells. A1 receptor signaling blocked bacterial binding by reducing the expression of platelet-activating factor receptor, a host protein used by S. pneumoniae to adhere to host cells. In vivo, A1 was required for control of pneumococcal pneumonia as inhibiting it resulted in increased host susceptibility. As S. pneumoniae remain a leading cause of community-acquired pneumonia in the elderly, we explored the role of A1 in the age-driven susceptibility to infection. We found no difference in A1 pulmonary expression in young versus old mice. Strikingly, triggering A1 signaling boosted host resistance of old mice to S. pneumoniae pulmonary infection. This study demonstrates a novel mechanism by which extracellular adenosine modulates resistance to lung infection by targeting bacterial-host interactions.
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http://dx.doi.org/10.1111/cmi.13141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980675PMC
February 2020

Prophage induction, but not production of phage particles, is required for lethal disease in a microbiome-replete murine model of enterohemorrhagic E. coli infection.

PLoS Pathog 2019 01 10;15(1):e1007494. Epub 2019 Jan 10.

Department of Molecular Biology and Microbiology at Tufts University School of Medicine, Boston, MA, United States of America.

Enterohemorrhagic Escherichia coli (EHEC) colonize intestinal epithelium by generating characteristic attaching and effacing (AE) lesions. They are lysogenized by prophage that encode Shiga toxin 2 (Stx2), which is responsible for severe clinical manifestations. As a lysogen, prophage genes leading to lytic growth and stx2 expression are repressed, whereas induction of the bacterial SOS response in response to DNA damage leads to lytic phage growth and Stx2 production both in vitro and in germ-free or streptomycin-treated mice. Some commensal bacteria diminish prophage induction and concomitant Stx2 production in vitro, whereas it has been proposed that phage-susceptible commensals may amplify Stx2 production by facilitating successive cycles of infection in vivo. We tested the role of phage induction in both Stx production and lethal disease in microbiome-replete mice, using our mouse model encompassing the murine pathogen Citrobacter rodentium lysogenized with the Stx2-encoding phage Φstx2dact. This strain generates EHEC-like AE lesions on the murine intestine and causes lethal Stx-mediated disease. We found that lethal mouse infection did not require that Φstx2dact infect or lysogenize commensal bacteria. In addition, we detected circularized phage genomes, potentially in the early stage of replication, in feces of infected mice, confirming that prophage induction occurs during infection of microbiota-replete mice. Further, C. rodentium (Φstx2dact) mutants that do not respond to DNA damage or express stx produced neither high levels of Stx2 in vitro or lethal infection in vivo, confirming that SOS induction and concomitant expression of phage-encoded stx genes are required for disease. In contrast, C. rodentium (Φstx2dact) mutants incapable of prophage genome excision or of packaging phage genomes retained the ability to produce Stx in vitro, as well as to cause lethal disease in mice. Thus, in a microbiome-replete EHEC infection model, lytic induction of Stx-encoding prophage is essential for lethal disease, but actual phage production is not.
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http://dx.doi.org/10.1371/journal.ppat.1007494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328086PMC
January 2019

Transporters MRP1 and MRP2 Regulate Opposing Inflammatory Signals To Control Transepithelial Neutrophil Migration during Streptococcus pneumoniae Lung Infection.

mSphere 2018 07 5;3(4). Epub 2018 Jul 5.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA

remains a source of morbidity and mortality in both developed and underdeveloped nations of the world. Disease can manifest as pneumonia, bacteremia, and meningitis, depending on the localization of infection. Interestingly, there is a correlation in experimental murine infections between the development of bacteremia and influx of neutrophils into the pulmonary lumen. Reduction of this neutrophil influx has been shown to improve survivability during infection. In this study, we use biotinylation and neutrophil transmigration and murine infection to identify a system in which two epithelium-localized ATP-binding cassette transporters, MRP1 and MRP2, have inverse activities dictating neutrophil transmigration into the lumen of infected mouse lungs. MRP1 effluxes an anti-inflammatory molecule that maintains homeostasis in uninfected contexts, thus reducing neutrophil infiltration. During inflammatory events, however, MRP1 decreases and MRP2 both increases and effluxes the proinflammatory eicosanoid hepoxilin A3. If we then decrease MRP2 activity during experimental murine infection with , we reduce both neutrophil infiltration and bacteremia, showing that MRP2 coordinates this activity in the lung. We conclude that MRP1 assists in depression of polymorphonuclear cell (PMN) migration by effluxing a molecule that inhibits the proinflammatory effects of MRP2 activity. is a Gram-positive bacterium that normally inhabits the human nasopharynx asymptomatically. However, it is also a major cause of pneumonia, bacteremia, and meningitis. The transition from pneumonia to bacteremia is critical, as patients that develop septicemia have ~20% mortality rates. Previous studies have shown that while neutrophils, a major bacterium-induced leukocyte, aid in elimination, they also contribute to pathology and may mediate the lung-to-blood passage of the bacteria. Herein, we show that epithelium-derived MRP1 and MRP2 efflux immunomodulatory agents that assist in controlling passage of neutrophils during infection and that limiting neutrophil infiltration produced less bacteremia and better survival during murine infection. The importance of our work is twofold: ours is the first to identify an MRP1/MRP2 axis of neutrophil control in the lung. The second is to provide possible therapeutic targets to reduce excess inflammation, thus reducing the chances of developing bacteremia during pneumococcal pneumonia.
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http://dx.doi.org/10.1128/mSphere.00303-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034076PMC
July 2018

Attaching-and-Effacing Pathogens Exploit Junction Regulatory Activities of N-WASP and SNX9 to Disrupt the Intestinal Barrier.

Cell Mol Gastroenterol Hepatol 2018 Mar 15;5(3):273-288. Epub 2017 Dec 15.

Division of Gastroenterology/Nutrition and Center for Inflammatory Bowel Disease Treatment and Research, Boston Children's Hospital, Boston, Massachusetts.

Background & Aims: Neural Wiskott-Aldrich Syndrome protein (N-WASP) is a key regulator of the actin cytoskeleton in epithelial tissues and is poised to mediate cytoskeletal-dependent aspects of apical junction complex (AJC) homeostasis. Attaching-and-effacing (AE) pathogens disrupt this homeostasis through translocation of the effector molecule early secreted antigenic target-6 (ESX)-1 secretion-associated protein F (EspF). Although the mechanisms underlying AJC disruption by EspF are unknown, EspF contains putative binding sites for N-WASP and the endocytic regulator sorting nexin 9 (SNX9). We hypothesized that N-WASP regulates AJC integrity and AE pathogens use EspF to induce junction disassembly through an N-WASP- and SNX9-dependent pathway.

Methods: We analyzed mice with intestine-specific N-WASP deletion and generated cell lines with N-WASP and SNX9 depletion for dynamic functional assays. We generated EPEC and strains complemented with EspF bearing point mutations abolishing N-WASP and SNX9 binding to investigate the requirement for these interactions.

Results: Mice lacking N-WASP in the intestinal epithelium showed spontaneously increased permeability, abnormal AJC morphology, and mislocalization of occludin. N-WASP depletion in epithelial cell lines led to impaired assembly and disassembly of tight junctions in response to changes in extracellular calcium. Cells lacking N-WASP or SNX9 supported actin pedestals and type III secretion, but were resistant to EPEC-induced AJC disassembly and loss of transepithelial resistance. We found that during in vivo infection with AE pathogens, EspF must bind both N-WASP and SNX9 to disrupt AJCs and induce intestinal barrier dysfunction.

Conclusions: Overall, these studies show that N-WASP critically regulates AJC homeostasis, and the AE pathogen effector EspF specifically exploits both N-WASP and SNX9 to disrupt intestinal barrier integrity during infection.
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http://dx.doi.org/10.1016/j.jcmgh.2017.11.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904039PMC
March 2018

Nasopharyngeal Exposure to Induces Extended Age-Dependent Protection against Pulmonary Infection Mediated by Antibodies and CD138 Cells.

J Immunol 2018 06 16;200(11):3739-3751. Epub 2018 Apr 16.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111;

commonly resides asymptomatically in the nasopharyngeal (NP) cavity of healthy individuals but can cause life-threatening pulmonary and systemic infections, particularly in the elderly. NP colonization results in a robust immune response that protects against invasive infections. However, the duration, mechanism, and cellular component of such responses are poorly understood. In this study, we found that repeated NP exposure of mice to TIGR4 strain results in pneumococcal-specific Ab responses that protect against lethal lung challenge. Abs were necessary and sufficient for protection because Ab-deficient μMT mice did not develop postexposure protection, only becoming resistant to lung infection after transfer of immune sera from NP-exposed mice. T cells contributed to immunity at the time of NP exposure, but neither CD4 nor CD8 T cells were required. The protective activity was detectable 20 wk after exposure and was maintained in irradiated mice, suggesting involvement of long-lived Ab-secreting cells (ASC), which are radioresistant and secrete Abs for extended periods of time in the absence of T cells or persistent Ag. CD138 bone marrow cells, likely corresponding to long-lived ASC, were sufficient to confer protection. NP exposure of aged mice failed to protect against subsequent lung infection despite eliciting a robust Ab response. Furthermore, transfer of CD138 bone marrow cells or sera from NP-exposed old mice failed to protect naive young mice. These findings suggest that NP exposure elicits extended protection against pneumococcal lung infection by generating long-lived CD138 ASC and that the protective efficacy of these responses declines with age.
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http://dx.doi.org/10.4049/jimmunol.1701065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047064PMC
June 2018

Plasticity in early immune evasion strategies of a bacterial pathogen.

Proc Natl Acad Sci U S A 2018 04 2;115(16):E3788-E3797. Epub 2018 Apr 2.

Department of Veterinary Medicine, University of Maryland, College Park, MD 20742;

is one of the few extracellular pathogens capable of establishing persistent infection in mammals. The mechanisms that sustain long-term survival of this bacterium are largely unknown. Here we report a unique innate immune evasion strategy of , orchestrated by a surface protein annotated as BBA57, through its modulation of multiple spirochete virulent determinants. BBA57 function is critical for early infection but largely redundant for later stages of spirochetal persistence, either in mammals or in ticks. The protein influences host IFN responses as well as suppresses multiple host microbicidal activities involving serum complement, neutrophils, and antimicrobial peptides. We also discovered a remarkable plasticity in BBA57-mediated spirochete immune evasion strategy because its loss, although resulting in near clearance of pathogens at the inoculum site, triggers nonheritable adaptive changes that exclude detectable nucleotide alterations in the genome but incorporate transcriptional reprograming events. Understanding the malleability in spirochetal immune evasion mechanisms that ensures their host persistence is critical for the development of novel therapeutic and preventive approaches to combat long-term infections like Lyme borreliosis.
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http://dx.doi.org/10.1073/pnas.1718595115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910839PMC
April 2018

Pearls collections: What we can learn about infectious disease and cancer.

PLoS Pathog 2018 03 29;14(3):e1006915. Epub 2018 Mar 29.

Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, United States of America.

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http://dx.doi.org/10.1371/journal.ppat.1006915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5875890PMC
March 2018

Characterization of Borrelia burgdorferi Binding to Mammalian Cells and Extracellular Matrix.

Methods Mol Biol 2018 ;1690:57-67

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA, 02111, USA.

Lyme disease Borreliae produces outer surface adhesins to confer bacterial attachment to the extracellular matrix (ECM) components on the surface of mammalian cells. Here, we describe protocols to characterize the activity and specificity of these adhesins by flow cytometry or measurement of the binding of radiolabeled spirochetes to immobilized ECM or mammalian cells.
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http://dx.doi.org/10.1007/978-1-4939-7383-5_5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6291642PMC
June 2018

Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival.

Cell Microbiol 2017 12 26;19(12). Epub 2017 Sep 26.

Department of Microbiology and Immunology, Center for Infectious Disease Research, Medical College of Wisconsin, Milwaukee, WI, USA.

Borrelia burgdorferi (Bb) is the causative agent of Lyme disease in the United States, a disease that can result in carditis, and chronic and debilitating arthritis and/or neurologic symptoms if left untreated. Bb survives in the midgut of the Ixodes scapularis tick, or within tissues of immunocompetent hosts. In the early stages of infection, the bacteria are present in the bloodstream where they must resist clearance by the innate immune system of the host. We have found a novel role for outer surface protein C (OspC) from B. burgdorferi and B. garinii in interactions with the complement component C4b and bloodstream survival in vivo. Our data show that OspC inhibits the classical and lectin complement pathways and competes with complement protein C2 for C4b binding. Resistance to complement is important for maintenance of the lifecycle of Bb, enabling survival of the pathogen within the host as well as in the midgut of a feeding tick when ospC expression is induced.
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http://dx.doi.org/10.1111/cmi.12786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680108PMC
December 2017

Cytosolic Phospholipase Aα Promotes Pulmonary Inflammation and Systemic Disease during Streptococcus pneumoniae Infection.

Infect Immun 2017 11 18;85(11). Epub 2017 Oct 18.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA

Pulmonary infection by is characterized by a robust alveolar infiltration of neutrophils (polymorphonuclear cells [PMNs]) that can promote systemic spread of the infection if not resolved. We previously showed that 12-lipoxygenase (12-LOX), which is required to generate the PMN chemoattractant hepoxilin A (HXA) from arachidonic acid (AA), promotes acute pulmonary inflammation and systemic infection after lung challenge with As phospholipase A (PLA) promotes the release of AA, we investigated the role of PLA in local and systemic disease during infection. The group IVA cytosolic isoform of PLA (cPLAα) was activated upon infection of cultured lung epithelial cells and was critical for AA release from membrane phospholipids. Pharmacological inhibition of this enzyme blocked -induced PMN transepithelial migration Genetic ablation of the cPLA isoform cPLAα dramatically reduced lung inflammation in mice upon high-dose pulmonary challenge with The cPLAα-deficient mice also suffered no bacteremia and survived a pulmonary challenge that was lethal to wild-type mice. Our data suggest that cPLAα plays a crucial role in eliciting pulmonary inflammation during pneumococcal infection and is required for lethal systemic infection following lung challenge.
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http://dx.doi.org/10.1128/IAI.00280-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5649009PMC
November 2017

The Alpha-Tocopherol Form of Vitamin E Boosts Elastase Activity of Human PMNs and Their Ability to Kill .

Front Cell Infect Microbiol 2017 3;7:161. Epub 2017 May 3.

Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts UniversityBoston MA, USA.

Despite the availability of vaccines, remains a leading cause of life-threatening infections, such as pneumonia, bacteremia and meningitis. Polymorphonuclear leukocytes (PMNs) are a key determinant of disease course, because optimal host defense requires an initial robust pulmonary PMN response to control bacterial numbers followed by modulation of this response later in infection. The elderly, who manifest a general decline in immune function and higher basal levels of inflammation, are at increased risk of developing pneumococcal pneumonia. Using an aged mouse infection model, we previously showed that oral supplementation with the alpha-tocopherol form of vitamin E (α-Toc) decreases pulmonary inflammation, in part by modulating neutrophil migration across lung epithelium into alveolar spaces, and reverses the age-associated decline in resistance to pneumococcal pneumonia. The objective of this study was to test the effect of α-Toc on the ability of neutrophils isolated from young (22-35 years) or elderly (65-69 years) individuals to migrate across epithelial cell monolayers in response to and to kill complement-opsonized pneumococci. We found that basal levels of pneumococcal-induced transepithelial migration by PMNs from young or elderly donors were indistinguishable, suggesting that the age-associated exacerbation of pulmonary inflammation is not due to intrinsic properties of PMNs of elderly individuals but rather may reflect the inflammatory milieu of the aged lung. Consistent with its anti-inflammatory activity, α-Toc treatment diminished PMN migration regardless of donor age. Unexpectedly, unlike previous studies showing poor killing of antibody-opsonized bacteria, we found that PMNs of elderly donors were more efficient at killing complement-opsonized bacteria than their younger counterparts. We also found that the heightened antimicrobial activity in PMNs from older donors correlated with increased activity of neutrophil elastase, a serine protease that is required to kill pneumococci. Notably, incubation with α-Toc increased PMN elastase activity from young donors and boosted their ability to kill complement-opsonized pneumococci. These findings demonstrate that α-Toc is a potent modulator of PMN responses and is a potential nutritional intervention to combat pneumococcal infection.
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http://dx.doi.org/10.3389/fcimb.2017.00161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413490PMC
December 2017

Synchronous Disease Kinetics in a Murine Model for Enterohemorrhagic Infection Using Food-Borne Inoculation.

Front Cell Infect Microbiol 2016 3;6:138. Epub 2016 Nov 3.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine Boston, MA, USA.

Upon colonization of the intestinal epithelium, the attaching and effacing (AE) pathogen Enterohemorrhagic (EHEC) effaces microvilli and forms pedestal-like structures beneath the adherent bacterium. The production of one of its virulence factors, the phage-encoded Shiga toxin (Stx) results in systemic disease, including the development of renal failure. Although EHEC does not productively infect conventional mice, EHEC infection can be modeled in mice utilizing a derivative of the natural murine AE pathogen (CR). Gavage of mice with CR(ΦStx), a lysogenized by a phage encoding an Stx variant with high potency in mice, features AE lesion formation on intestinal epithelium and Stx-mediated systemic disease, including renal damage. This model is somewhat limited by mouse-to-mouse variation in the course of disease, with the time to severe morbidity (and required euthanasia) varying by as many as 5 days, a feature that limits pathological analysis at defined stages of disease. In the current study, we altered and optimized the preparation, dose, and mode of delivery of CR(ΦStx), using food-borne route of infection to generate highly synchronous disease model. We found that food-borne inoculation of as few as 3 × 10 CR(ΦStx) resulted in productive colonization and severe systemic disease. Upon inoculation of 1 × 10 bacteria, the majority of infected animals suffered weight loss beginning 5 days post-infection and all required euthanasia on day 6 or 7. This enhanced murine model for EHEC infection should facilitate characterization of the pathology associated with specific phases of Stx-mediated disease.
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http://dx.doi.org/10.3389/fcimb.2016.00138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5093121PMC
September 2017

A Novel Zak Knockout Mouse with a Defective Ribotoxic Stress Response.

Toxins (Basel) 2016 09 2;8(9). Epub 2016 Sep 2.

Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA 02111, USA.

Ricin activates the proinflammatory ribotoxic stress response through the mitogen activated protein 3 kinase (MAP3K) ZAK, resulting in activation of mitogen activated protein kinases (MAPKs) p38 and JNK1/2. We had a novel zak-/- mouse generated to study the role of ZAK signaling in vivo during ricin intoxication. To characterize this murine strain, we intoxicated zak-/- and zak+/+ bone marrow-derived murine macrophages with ricin, measured p38 and JNK1/2 activation by Western blot, and measured zak, c-jun, and cxcl-1 expression by qRT-PCR. To determine whether zak-/- mice differed from wild-type mice in their in vivo response to ricin, we performed oral ricin intoxication experiments with zak+/+ and zak-/- mice, using blinded histopathology scoring of duodenal tissue sections to determine differences in tissue damage. Unlike macrophages derived from zak+/+ mice, those derived from the novel zak-/- strain fail to activate p38 and JNK1/2 and have decreased c-jun and cxcl-1 expression following ricin intoxication. Furthermore, compared with zak+/+ mice, zak-/- mice have decreased duodenal damage following in vivo ricin challenge. zak-/- mice demonstrate a distinct ribotoxic stress-associated phenotype in response to ricin and therefore provide a new animal model for in vivo studies of ZAK signaling.
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http://dx.doi.org/10.3390/toxins8090259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5037485PMC
September 2016

Activation of the Classical Mitogen-Activated Protein Kinases Is Part of the Shiga Toxin-Induced Ribotoxic Stress Response and May Contribute to Shiga Toxin-Induced Inflammation.

Infect Immun 2016 01 19;84(1):138-48. Epub 2015 Oct 19.

Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, Massachusetts, USA.

Infection with enterohemorrhagic Escherichia coli (EHEC) can result in severe disease, including hemorrhagic colitis and the hemolytic uremic syndrome. Shiga toxins (Stx) are the key EHEC virulence determinant contributing to severe disease. Despite inhibiting protein synthesis, Shiga toxins paradoxically induce the expression of proinflammatory cytokines from various cell types in vitro, including intestinal epithelial cells (IECs). This effect is mediated in large part by the ribotoxic stress response (RSR). The Shiga toxin-induced RSR is known to involve the activation of the stress-activated protein kinases (SAPKs) p38 and JNK. In some cell types, Stx also can induce the classical mitogen-activated protein kinases (MAPKs) or ERK1/2, but the mechanism(s) by which this activation occurs is unknown. In this study, we investigated the mechanism by which Stx activates ERK1/2s in IECs and the contribution of ERK1/2 activation to interleukin-8 (IL-8) expression. We demonstrate that Stx1 activates ERK1/2 in a biphasic manner: the first phase occurs in response to StxB1 subunit, while the second phase requires StxA1 subunit activity. We show that the A subunit-dependent ERK1/2 activation is mediated through ZAK-dependent signaling, and inhibition of ERK1/2 activation via the MEK1/2 inhibitors U0126 and PD98059 results in decreased Stx1-mediated IL-8 mRNA. Finally, we demonstrate that ERK1/2 are activated in vivo in the colon of Stx2-intoxicated infant rabbits, a model in which Stx2 induces a primarily neutrophilic inflammatory response. Together, our data support a role for ERK1/2 activation in the development of Stx-mediated intestinal inflammation.
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http://dx.doi.org/10.1128/IAI.00977-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694011PMC
January 2016

Extracellular Adenosine Protects against Streptococcus pneumoniae Lung Infection by Regulating Pulmonary Neutrophil Recruitment.

PLoS Pathog 2015 Aug 27;11(8):e1005126. Epub 2015 Aug 27.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

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 lung infection. Moreover, adenosine diminished PMN movement across endothelial monolayers in vitro, and although inhibition or deficiency of CD73 had no discernible impact on PMN recruitment within the first 6 hours after intratracheal inoculation of mice, these measures enhanced PMN numbers in the pulmonary interstitium after 18 hours of infection, culminating in dramatically elevated numbers of pulmonary PMNs at three days post-infection. When assessed at this time point, CD73-/- mice displayed increased levels of cellular factors that promote leukocyte migration, such as CXCL2 chemokine in the murine lung, as well as CXCR2 and β-2 integrin on the surface of pulmonary PMNs. The enhanced pneumococcal susceptibility of CD73-/- mice was significantly reversed by PMN depletion following infection, suggesting that EAD-mediated resistance is largely mediated by its effects on PMNs. Finally, CD73-inhibition diminished the ability of PMNs to kill pneumococci in vitro, suggesting that EAD alters both the recruitment and bacteriocidal function of PMNs. The EAD-pathway may provide a therapeutic target for regulating potentially harmful inflammatory host responses during Gram-positive bacterial pneumonia.
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http://dx.doi.org/10.1371/journal.ppat.1005126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4552087PMC
August 2015

Middle region of the Borrelia burgdorferi surface-located protein 1 (Lmp1) interacts with host chondroitin-6-sulfate and independently facilitates infection.

Cell Microbiol 2016 Jan 31;18(1):97-110. Epub 2015 Aug 31.

Department of Veterinary Medicine, University of Maryland, College Park and Virginia-Maryland Regional College of Veterinary Medicine, College Park, MD, 20742, USA.

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.
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http://dx.doi.org/10.1111/cmi.12487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718918PMC
January 2016

Host cell heparan sulfate glycosaminoglycans are ligands for OspF-related proteins of the Lyme disease spirochete.

Cell Microbiol 2015 Oct 13;17(10):1464-76. Epub 2015 May 13.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave, Boston, MA, 02111, USA.

Borrelia burgdorferi, the agent of Lyme disease, spreads from the site of the tick bite to tissues such as heart, joints and the nervous tissues. Host glycosaminoglycans, 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 non-adherent B. burgdorferi strain, ErpG promotes heparan sulfate-mediated bacterial attachment to the glial but not the endothelial, synovial or respiratory epithelial cells. Six other OspF-related proteins were capable of binding heparan sulfate, whereas representative OspE-related and Elp proteins lacked this activity. These results indicate that OspF-related proteins are heparan sulfate-binding adhesins, at least one of which promotes bacterial attachment to glial cells.
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http://dx.doi.org/10.1111/cmi.12448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4583806PMC
October 2015

Src-dependent tyrosine phosphorylation of non-muscle myosin heavy chain-IIA restricts Listeria monocytogenes cellular infection.

J Biol Chem 2015 Mar 29;290(13):8383-95. Epub 2015 Jan 29.

From the Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200 Porto, Portugal, the Group of Molecular Microbiology, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal,

Bacterial pathogens often interfere with host tyrosine phosphorylation cascades to control host responses and cause infection. Given the role of tyrosine phosphorylation events in different human infections and our previous results showing the activation of the tyrosine kinase Src upon incubation of cells with Listeria monocytogenes, we searched for novel host proteins undergoing tyrosine phosphorylation upon L. monocytogenes infection. We identify the heavy chain of the non-muscle myosin IIA (NMHC-IIA) as being phosphorylated in a specific tyrosine residue in response to L. monocytogenes infection. We characterize this novel post-translational modification event and show that, upon L. monocytogenes infection, Src phosphorylates NMHC-IIA in a previously uncharacterized tyrosine residue (Tyr-158) located in its motor domain near the ATP-binding site. In addition, we found that other intracellular and extracellular bacterial pathogens trigger NMHC-IIA tyrosine phosphorylation. We demonstrate that NMHC-IIA limits intracellular levels of L. monocytogenes, and this is dependent on the phosphorylation of Tyr-158. Our data suggest a novel mechanism of regulation of NMHC-IIA activity relying on the phosphorylation of Tyr-158 by Src.
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http://dx.doi.org/10.1074/jbc.M114.591313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375491PMC
March 2015

Integrin binding by Borrelia burgdorferi P66 facilitates dissemination but is not required for infectivity.

Cell Microbiol 2015 Jul 16;17(7):1021-36. Epub 2015 Feb 16.

Graduate Program in Microbiology, Immunology, and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA.

P66, a Borrelia burgdorferi surface protein with porin and integrin-binding activities, is essential for murine infection. The role of P66 integrin-binding activity in B. burgdorferi infection was investigated and found to affect transendothelial migration. The role of integrin binding, specifically, was tested by mutation of two amino acids (D205A,D207A) or deletion of seven amino acids (Del202-208). Neither change affected surface localization or channel-forming activity of P66, but both significantly reduced binding to αv β3 . Integrin-binding deficient B. burgdorferi strains caused disseminated infection in mice at 4 weeks post-subcutaneous inoculation, but bacterial burdens were significantly reduced in some tissues. Following intravenous inoculation, the Del202-208 bacteria were below the limit of detection in all tissues assessed at 2 weeks post-inoculation, but bacterial burdens recovered to wild-type levels at 4 weeks post-inoculation. The delay in tissue colonization correlated with reduced migration of the Del202-208 strains across microvascular endothelial cells, similar to Δp66 bacteria. These results indicate that integrin binding by P66 is important to efficient dissemination of B. burgdorferi, which is critical to its ability to cause disease manifestations in incidental hosts and to its maintenance in the enzootic cycle.
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http://dx.doi.org/10.1111/cmi.12418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478124PMC
July 2015

The α-tocopherol form of vitamin E reverses age-associated susceptibility to streptococcus pneumoniae lung infection by modulating pulmonary neutrophil recruitment.

J Immunol 2015 Feb 15;194(3):1090-9. Epub 2014 Dec 15.

Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston MA 02114; and

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 infection. This α-Toc-induced resistance to pneumococcal challenge was associated with a 2-fold fewer pulmonary neutrophils, a level comparable to S. pneumoniae-challenged, conventionally fed young mice. α-Toc directly inhibited neutrophil egress across epithelial cell monolayers in vitro in response to pneumococci or hepoxilin-A3, an eicosanoid required for pneumococcus-elicited neutrophil trans-epithelial migration. α-Toc altered expression of multiple epithelial and neutrophil adhesion molecules involved in migration, including CD55, CD47, CD18/CD11b, and ICAM-1. These findings suggest that α-Toc enhances resistance of aged mice to bacterial pneumonia by modulating the innate immune response, a finding that has potential clinical significance in combating infection in aged individuals through nutritional intervention.
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http://dx.doi.org/10.4049/jimmunol.1402401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834212PMC
February 2015

Glycosaminoglycan binding by Borrelia burgdorferi adhesin BBK32 specifically and uniquely promotes joint colonization.

Cell Microbiol 2015 Jun 24;17(6):860-75. Epub 2015 Jan 24.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave, Boston, MA, 02111, USA.

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 infection. Intravenous inoculation of mice with a high-passage non-infectious B. burgdorferi strain that produced wild-type BBK32 or BBK32 mutants defective for GAG or fibronectin binding, revealed that only GAG-binding activity was required for significant localization to joints at 60 min post-infection. An otherwise infectious B. burgdorferi strain producing BBK32 specifically deficient in fibronectin binding was fully capable of both skin and joint colonization in the murine model, whereas a strain producing BBK32 selectively attenuated for GAG binding colonized the inoculation site but not knee or tibiotarsus joints. Thus, the BBK32 fibronectin- and GAG-binding activities are separable in vivo, and BBK32-mediated GAG binding, but not fibronectin binding, contributes to joint colonization.
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http://dx.doi.org/10.1111/cmi.12407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4437914PMC
June 2015

Effects of Shiga toxin type 2 on a bioengineered three-dimensional model of human renal tissue.

Infect Immun 2015 Jan 13;83(1):28-38. Epub 2014 Oct 13.

Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, USA

Shiga toxins (Stx) are a family of cytotoxic proteins that can cause hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy, following infections by Shiga toxin-producing Escherichia coli (STEC). Renal failure is a key feature of HUS and a major cause of childhood renal failure worldwide. There are currently no specific therapies for STEC-associated HUS, and the mechanism of Stx-induced renal injury is not well understood primarily due to a lack of fully representative animal models and an inability to monitor disease progression on a molecular or cellular level in humans at early stages. Three-dimensional (3D) tissue models have been shown to be more in vivo-like in their phenotype and physiology than 2D cultures for numerous disease models, including cancer and polycystic kidney disease. It is unknown whether exposure of a 3D renal tissue model to Stx will yield a more in vivo-like response than 2D cell culture. In this study, we characterized Stx2-mediated cytotoxicity in a bioengineered 3D human renal tissue model previously shown to be a predictor of drug-induced nephrotoxicity and compared its response to Stx2 exposure in 2D cell culture. Our results demonstrate that although many mechanistic aspects of cytotoxicity were similar between 3D and 2D, treatment of the 3D tissues with Stx resulted in an elevated secretion of the kidney injury marker 1 (Kim-1) and the cytokine interleukin-8 compared to the 2D cell cultures. This study represents the first application of 3D tissues for the study of Stx-mediated kidney injury.
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http://dx.doi.org/10.1128/IAI.02143-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288879PMC
January 2015