Jenifer Coburn

The emergence of Lyme disease

Since its identification nearly 30 years ago, Lyme disease has continued to spread, and there have been increasing numbers of cases in the northeastern and north central US. The Lyme disease agent, Borrelia burgdorferi, causes infection by migration through tissues, adhesion to host cells, and evasion of immune clearance. Both innate and adaptive immune responses, especially macrophage- and antibody-mediated killing, are required for optimal control of the infection and spirochetal eradication. Ecological conditions favorable to the disease, and the challenge of prevention, predict that Lyme disease will be a continuing public health concern.

Leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune responses

Leptospirosis, the most widespread zoonosis in the world, is an emerging public health problem, particularly in large urban centers of developing countries. Several pathogenic species of the genus Leptospira can cause a wide range of clinical manifestations, from a mild, flu-like illness to a severe disease form characterized by multiorgan system complications leading to death. However, the mechanisms of pathogenesis of Leptospira are largely unknown. This article will address the animal models of acute and chronic leptospire infections, and the recent developments in the genetic manipulation of the bacteria, which facilitate the identification of virulence factors involved in pathogenesis and the assessment of their potential values in the control and prevention of leptospirosis.

Characterization of a candidate Borrelia burgdorferi beta3-chain integrin ligand identified using a phage display library.

The spirochaetal agents of Lyme disease, Borrelia burgdorferi (sensu lato) bind to integrins αIIbβ3, αvβ3 and α5β1 in purified form and on the surfaces of human cells. Using a phage display library of B. burgdorferi (sensu stricto) DNA, a candidate ligand for β3‐chain integrins was identified. The native B. burgdorferi protein, termed p66, is known to be recognized by human Lyme disease patient sera and to be expressed on the surface of the spirochaete. We show here that recombinant p66 binds specifically to β3‐chain integrins and inhibits attachment of intact B. burgdorferi to the same integrins. When expressed on the surface of Escherichia coli, this protein increases the attachment of E. coli to a transfected cell line that expresses αvβ3, but not to the parental cell line, which expresses no β3‐chain integrins. Localization of p66 on the surface of B. burgdorferi, the ability of recombinant forms of the protein to bind to β3‐chain integrins and the fact that p66 and B. burgdorferi bind to β3‐chain integrins in a mutually exclusive manner make p66 an attractive candidate bacterial ligand for integrins αIIbβ3 and αvβ3.

Solving a sticky problem: new genetic approaches to host cell adhesion by the Lyme disease spirochete

The Lyme disease spirochetes, comprised of at least three closely related species, Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii, are fascinating and enigmatic bacterial pathogens. They are maintained by tick‐mediated transmission between mammalian hosts, usually small rodents. The ability of these bacteria, which have relatively small genomes, to survive and disseminate in both an immunocompetent mammal and in an arthropod vector suggests that they have evolved elegant and indispensable strategies for interacting with their hosts. Recognition of specific mammalian and tick tissues is likely to be essential for successful completion of the enzootic life cycle but, given the historical difficulties in genetic manipulation of these organisms, characterization of factors promoting cell adhesion has until recently largely been confined to either the manipulation of host cells or the analysis of potential bacterial ligands in the form of recombinant proteins. These studies have led to the identification of several mammalian receptors for Lyme disease spirochetes, including glycosaminoglycans, decorin, fibronectin and integrins, as well as a tick receptor for the bacterium, and also candidate cognate bacterial ligands. Recent advances in our ability to genetically manipulate Lyme disease spirochetes, particularly B. burgdorferi, are now providing us with firm evidence that these ligands indeed do promote bacterial adherence to host cells, and with new insights into the roles of these multifacted Borrelia–host cell interactions during mammalian and arthropod infection.

Targeted mutation of the outer membrane protein P66 disrupts attachment of the Lyme disease agent, Borrelia burgdorferi, to integrin αvβ3

Borrelia burgdorferi, the agent of Lyme disease, expresses several adhesion molecules that are probably required for initial establishment of infection in mammalian hosts, and for colonization of various tissues within the host. The B. burgdorferi outer membrane protein P66 was previously identified as a ligand for β3-chain integrins by using a variety of biochemical approaches. Although the earlier data suggested that P66 is an adhesin that mediates B. burgdorferi attachment to β3-chain integrins, lack of genetic systems in B. burgdorferi precluded definitive demonstration of a role for P66 in β3 integrin attachment by intact borreliae. Recent advances in the genetic manipulation of B. burgdorferi have now made possible the targeted disruption of the p66 gene. Mutants in p66 show dramatically reduced attachment to integrin αvβ3. This is, to our knowledge, the first description of the targeted disruption of a candidate B. burgdorferi virulence factor with a known biochemical function that can be quantified, and demonstrates the importance of B. burgdorferi P66 in the attachment of this pathogenic spirochete to a human cell-surface receptor.

Borrelia burgdorferi adhesins identified using in vivo phage display

Borrelia burgdorferi, the agent of Lyme disease, disseminates from the site of deposition by Ixodes ticks to cause systemic infection. Dissemination occurs through the circulation and through tissue matrices, but the B. burgdorferi molecules that mediate interactions with the endothelium in vivo have not yet been identified. In vivo selection of filamentous phage expressing B. burgdorferi protein fragments on the phage surface identified several new candidate adhesins, and verified the activity of one adhesin that had been previously characterized in vitro. P66, a B. burgdorferi ligand for β3‐chain integrins, OspC, a protein that is essential for the establishment of infection in mammals, and Vls, a protein that undergoes antigenic variation in the mammal, were all selected for binding to the murine endothelium in vivo. Additional B. burgdorferi proteins for which no functions have been identified, including all four members of the OspF family and BmpD, were identified as candidate adhesins. The use of in vivo phage display is one approach to the identification of adhesins in pathogenic bacteria that are not easily grown in the laboratory, or for which genetic manipulations are not straightforward.

Borrelia burgdorferi BBB07 Interaction With Integrin α3β1 Stimulates Production of Pro-Inflammatory Mediators in Primary Human Chondrocytes

Borrelia burgdorferi, the causative agent of Lyme disease, activates multiple signalling pathways leading to induction of pro‐inflammatory mediators at sites of inflammation. Binding of B. burgdorferi to integrin α3β1 on human chondrocytes activates signalling leading to release of several pro‐inflammatory mediators, but the B. burgdorferi protein that binds integrin α3β1 and elicits this response has remained unknown. A search of the B. burgdorferi genome for a canonical integrin binding motif, the RGD (Arg–Gly–Asp) tripeptide, revealed several candidate ligands for integrins. In this study we show that one of these candidates, BBB07, binds to integrin α3β1 and inhibits attachment of intact B. burgdorferi to the same integrin. BBB07 is expressed during murine infection as demonstrated by recognition by infected mouse sera. Recombinant purified BBB07 induces pro‐inflammatory mediators in primary human chondrocyte cells by interaction with integrin α3β1. This interaction is specific, as P66, another integrin ligand of B. burgdorferi, does not activate signalling through α3β1. In summary, we have identified a B. burgdorferi protein, BBB07, that interacts with integrin α3β1 and stimulates production of pro‐inflammatory mediators in primary human chondrocyte cells.

Identification of a TLR-Independent Pathway for Borrelia burgdorferi-Induced Expression of Matrix Metalloproteinases and Inflammatory Mediators through Binding to Integrin α3β1

Borrelia burgdorferi stimulates a robust inflammatory response at sites of localization. Binding of borrelial lipoproteins to TLR-2 is one pathway important in the host response to B. burgdorferi. However, while TLR-2 is clearly important in control of infection, inflammation is actually worsened in the absence of TLR-2 or the shared TLR adapter molecule, MyD88, suggesting that there are alternative pathways regulating inflammation. Integrins are cell surface receptors that play an important role in cell to cell communications and that can activate inflammatory signaling pathways. In this study, we report for the first time that B. burgdorferi binds to integrin α3β1 and that binding of B. burgdorferi to this integrin results in induction of proinflammatory cytokines, chemokines, and end-effector molecules such as matrix metalloproteinases in primary human chondrocyte cells. Expression of these same molecules is not affected by the absence of MyD88 in murine articular cartilage, suggesting that the two pathways act independently in activating host inflammatory responses to B. burgdorferi. B. burgdorferi-induced α3 signaling is mediated by JNK, but not p38 MAPK. In summary, we have identified a new host receptor for B. burgdorferi, integrin α3β1; binding of B. burgdorferi to integrin α3β1 results in the release of inflammatory mediators and is proposed as a TLR-independent pathway for activation of the innate immune response by the organism.

Characterization of Pseudomonas aeruginosa Exoenzyme S as a Bifunctional Enzyme in J774A.1 Macrophages

ABSTRACT Pseudomonas aeruginosa exoenzyme S (ExoS) is a type III secretion (TTS) effector, which includes both a GTPase-activating protein (GAP) activity toward the Rho family of low-molecular-weight G (LMWG) proteins and an ADP-ribosyltransferase (ADPRT) activity that targets LMWG proteins in the Ras, Rab, and Rho families. The coordinate function of both activities of ExoS in J774A.1 macrophages was assessed by using P. aeruginosa strains expressing and translocating wild-type ExoS or ExoS defective in GAP and/or ADPRT activity. Distinct and coordinated functions were identified for both domains. The GAP activity was required for the antiphagocytic effect of ExoS and was linked to interference of lamellopodium and membrane ruffle formation. Alternatively, the ADPRT activity of ExoS altered cellular adherence and morphology and was linked to effects on filopodium formation. The cellular mechanism of ExoS GAP activity included an inactivation of Rac1 function, as determined in p21-activated kinase 1-glutathione S-transferase (GST) pull-down assays. The ADPRT activity of ExoS targeted Ras and RalA but not Rab or Rho proteins, and Ral binding protein 1-GST pull-down assays identified an effect of ExoS ADPRT activity on RalA activation. The results from these studies confirm the bifunctional nature of ExoS activity within macrophages when translocated by TTS.

Pseudomonas aeruginosa Exoenzyme S

Pseudomonas aeruginosa is an opportunistic pathogen that secretes a number of potential virulence factors. Two of these, exotoxin A and exoenzyme S, catalyze the transfer of the ADP-ribose moiety of NAD to proteins in eukaryotic cells. Exotoxin A catalyzes the ADP-ribosylation of elongation factor 2 (EF-2), leading to disruption of protein synthesis. It has been well characterized and is reviewed elsewhere in this volume. Exoenzyme S has been less thoroughly studied, but several lines of evidence suggest that it might play a role in pathogenesis. In preliminary experiments exoenzyme S appeared to be unselective in choice of substrate proteins, but recent work has shown that it preferentially ADP-ribosylates several of the low-molecular weight GTP-binding proteins. Furthermore, like cholera toxin, exoenzyme S requires a eukaryotic protein for enzymic activity.