Sarah Veloso Nogueira

Paracoccidioides brasiliensis enolase is a surface protein that binds plasminogen and mediates interaction of yeast forms with host cells.

ABSTRACT Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.

A Surface Enolase Participates in Borrelia burgdorferi-Plasminogen Interaction and Contributes to Pathogen Survival within Feeding Ticks

ABSTRACT Borrelia burgdorferi, a tick-borne bacterial pathogen, causes a disseminated infection involving multiple organs known as Lyme disease. Surface proteins can directly participate in microbial virulence by facilitating pathogen dissemination via interaction with host factors. We show here that a fraction of the B. burgdorferi chromosomal gene product BB0337, annotated as enolase or phosphopyruvate dehydratase, is associated with spirochete outer membrane and is surface exposed. B. burgdorferi enolase, either in a recombinant form or as a membrane-bound native antigen, displays enzymatic activities intrinsic to the glycolytic pathway. However, the protein also interacts with host plasminogen, potentially leading to its activation and resulting in B. burgdorferi-induced fibrinolysis. As expected, enolase displayed consistent expression in vivo, however, with a variable temporal and spatial expression during spirochete infection in mice and ticks. Despite an extracellular exposure of the antigen and a potential role in host-pathogen interaction, active immunization of mice with recombinant enolase failed to evoke protective immunity against subsequent B. burgdorferi infection. In contrast, enolase immunization of murine hosts significantly reduced the acquisition of spirochetes by feeding ticks, suggesting that the protein could have a stage-specific role in B. burgdorferi survival in the feeding vector. Strategies to interfere with the function of surface enolase could contribute to the development of novel preventive measures to interrupt the spirochete infection cycle and reduce the incidences of Lyme disease.

Recombinant BCG: Innovations on an Old Vaccine. Scope of BCG Strains and Strategies to Improve Long-Lasting Memory

Bacille Calmette–Guérin (BCG), an attenuated vaccine derived from Mycobacterium bovis, is the current vaccine of choice against tuberculosis (TB). Despite its protection against active TB in children, BCG has failed to protect adults against TB infection and active disease development, especially in developing countries where the disease is endemic. Currently, there is a significant effort toward the development of a new TB vaccine. This review article aims to address publications on recombinant BCG (rBCG) published in the last 5 years, to highlight the strategies used to develop rBCG, with a focus on the criteria used to improve immunological memory and protection compared with BCG. The literature review was done in April 2013, using the key words TB, rBCG vaccine, and memory. This review discusses the BCG strains and strategies currently used for the modification of BCG, including: overexpression of Mycobacterium tuberculosis (Mtb) immunodominant antigens already present in BCG; gene insertion of immunodominant antigens from Mtb absent in the BCG vaccine; combination of introduction and overexpression of genes that are lost during the attenuation process of BCG; BCG modifications for the induction of CD8+ T-cell immune responses and cytokines expressing rBCG. Among the vaccines discussed, VPM1002, also called rBCGΔureC:hly, is currently in human clinical trials. Much progress has been made in the effort to improve BCG, with some promising candidates, but considerable work is still required to address functional long-lasting memory.

Leptospira interrogans enolase is secreted extracellularly and interacts with plasminogen

Leptospira interrogans is the agent for leptospirosis, an important zoonosis in humans and animals across the globe. Surface proteins of invading pathogens, such as L. interrogans, are thought to be responsible for successful microbial persistence in vivo via interaction with specific host components. In particular, a number of invasive infectious agents exploit host proteolytic pathways, such as one involving plasminogen (Pg), which aid in efficient pathogen dissemination within the host. Here we show that L. interrogans serovar Lai binds host Pg and that the leptospiral gene product LA1951, annotated as enolase, is involved in this interaction. Interestingly, unlike in related pathogenic Spirochetes, such as Borrelia burgdorferi, LA1951 is not readily detectable in the L. interrogans outer membrane. We show that the antigen is indeed secreted extracellularly; however, it can reassociate with the pathogen surface, where it displays Pg-binding and measurable enzymatic activity. Hamsters infected with L. interrogans also develop readily detectable antibody responses against enolase. Taken together, our results suggest that the L. interrogans enolase has evolved to play a role in pathogen interaction with host molecules, which may contribute to the pathogenesis of leptospirosis.

A New Recombinant BCG Vaccine Induces Specific Th17 and Th1 Effector Cells with Higher Protective Efficacy against Tuberculosis

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that is a major public health problem. The vaccine used for TB prevention is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which provides variable efficacy in protecting against pulmonary TB among adults. Consequently, several groups have pursued the development of a new vaccine with a superior protective capacity to that of BCG. Here we constructed a new recombinant BCG (rBCG) vaccine expressing a fusion protein (CMX) composed of immune dominant epitopes from Ag85C, MPT51, and HspX and evaluated its immunogenicity and protection in a murine model of infection. The stability of the vaccine in vivo was maintained for up to 20 days post-vaccination. rBCG-CMX was efficiently phagocytized by peritoneal macrophages and induced nitric oxide (NO) production. Following mouse immunization, this vaccine induced a specific immune response in cells from lungs and spleen to the fusion protein and to each of the component recombinant proteins by themselves. Vaccinated mice presented higher amounts of Th1, Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of lung lesions caused by challenge with Mtb as well as the lung bacterial load. In addition, when this vaccine was used in a prime-boost strategy together with rCMX, the lung bacterial load was lower than the result observed by BCG vaccination. This study describes the creation of a new promising vaccine for TB that we hope will be used in further studies to address its safety before proceeding to clinical trials.

Characterization of alpha-enolase as an interferon-alpha 2 alpha 1 regulated gene.

Interferons (IFNs) are multifunctional cytokines that after binding to the cell surface receptor induce the expression of a large number of genes, which in turn, mediate many biological processes including host defense, cell growth control, signaling, and metabolism. Here we show that IFN-alpha activates the mitogen-activated protein kinases (MAPK) ERK1/2 and the transcription factor CREB/ATF-1, which lead to the alpha-enolase (alpha-ENO) gene expression in fibroblasts. Alpha-ENO mRNA accumulation was apparent 6 h post-IFN stimulation and required both de novo protein synthesis and active gene transcription, which is typical of a secondary response gene. Alpha-ENO expression does not appear to be restricted to fibroblasts, since it was equally verified in peripheral blood mononuclear cells (PBMC). Furthermore, IFN-alpha stimulates the expression of the primary response genes c-fos and egr-1, which was followed by an increase in DNA binding activity of c-FOS and EGR-1 proteins, as verified by shift assays using the cis-acting elements AP-1 and EGR-1 localized at the alpha-ENO promoter. Finally, we also demonstrated that IFN treatment of PBMC cause an increase in both, alpha-ENO expression on the cell surface and plasmin generation followed addition of exogenous plasminogen.