The use of Lactic Acid Bacteria (LAB), such as Lactococcus lactis (LL), as DNA delivery vehicles represents an interesting strategy as they are regarded as safe. Wild type (wt) LL or recombinant invasive LL, were able to trigger DNA expression by epithelial cells both in vitro and in vivo. However, important information about how LL can transfer DNA plasmids is still missing. Therefore, we decided to construct a new recombinant invasive LL strain expressing mutated Internalin A (mInlA) from the pathogen Listeria monocytogenes to understand the manner by which the DNA is transferred to mammalian cells. mInlA expression was detected by FACS analysis and LL-mInlA strain showed to be more invasive than the wt strain after co-incubation assays with non-confluent or polarized intestinal epithelial cells (IECs). Confocal microscopy confirmed the invasive status of LL-mInlA which demonstrated to deliver more efficiently the eukaryotic expression vector coding the allergen β-lactoglobulin, pValac:BLG, in vitro to IECs and to dendritic cells (DCs). LL-mInlA was also capable to transfer pValac:BLG to DCs across a monolayer of differentiated IECs. In vivo, invasive lactococci tended to increase the number of mice expressing BLG. Moreover, noninvasive or invasive LL-mInlA stimulated the secretion of the pro-inflammatory cytokine IL-12 in DCs and, in vivo, after oral or intranasal immunization trials, non-invasive LL polarized the immune response more in the type 1 direction while invasive LL generated a Th2-type response in immunized animals. All these data gives new insights on the mechanism of lactococci uptake for delivery of therapeutics.

Made available in DSpace on 2014-08-20T13:33:00Z (GMT). No. of bitstreams: 1 tese_marcelo_mendonca.PDF: 4204978 bytes, checksum: e41a9490cdb350a5e7add8e129afdd82 (MD5) Previous issue date: 2011-12-01 / The conventional methods used to detect the Listeria monocytogenes in foods are laborious and expensive, requiring several days for final identification. Monoclonal antibody (MAb) based immunoassays are highly specific and rapid to perform, especially when MAbs are raised to conserved virulence factors in the pathogen. Among diverse virulence factors of L. monocytogenes, the surface protein internalin A (InlA) is one of the most well-known and characterized protein, being an excellent target as it is highly exposed on the surface and exclusive of pathogenic species. In this work we report the production, characterization and use of a panel of MAbs against InlA (2D12, 3B7, 4E4), and a MAb (3F8) which specifically recognizes all bacteria belonging the genus Listeria. MAbs were produced by the immunization of BALB/c mice with a recombinant InlA together with heat killed L. monocytogenes. The MAbs produced showed excellent reativities by indirect ELISA, Western blot and immunofluorescence. A Cy5 conjugated anti-InlA MAb-2D12 was used as detection antibody for L. monocytogenes in a sandwich-like fiber optic immunoassay. Using MAb-2D12 as capture antibody on the waveguides, the limit of detection was ~3 x 102 CFU.mL-1, and when MAb-3F8 was used for capture the limit of detection was ~1 x 105 CFU.mL-1. Furthermore, MAbs 2D12 and 3F8 were used to coat paramagnetic beads and tested in the immunomagnetic separation (IMS) of L. monocytogenes from pure cultures, and artificially contaminated cheeses and hotdogs. After IMS capture, bacteria were released from the beads, used in the fiber optic assay or plated on agar for counting. In parallel, the capture of L. monocytogenes was confirmed by real-time qPCR and light-scattering technology (BARDOT). Using IMS to concentrate and separate L. monocytogenes, followed by a fiber optic platform, it was possible to detect in less than 22 h, approximately 40 CFU/g of L. monocytogenesi, even in the presence of L. innocua in cheese and hot dogs artificially contaminated. In addition, using mass spectrometry (MALDI-TOF-MS) the protein to which MAb-3F8 binds, was identified as fructose 1,6-bisphosphate aldolase (FBA). The results presented in this work indicate that using both systems together, the IMS and fiber optic immunosensor, were more reliable and faster, and could be applied in the routinely for detection of L. monocytogenes in food. Moreover, both MAbs have the potential to useful in others biosensor platforms, as well as in other detection and functionality immunoassays for InlA and FBA in Listeria. / Os métodos convencionais empregados para detecção de Listeria monocytogenes em alimentos são laboriosos e onerosos, requerendo vários dias para sua identificação final. A utilização de anticorpos monoclonais (MAbs) em imunoensaios para detecção rápida de bactérias tem como vantagem a alta especificidade e rapidez, principalmente quando direcionados para fatores de virulência conservados. Dentre os diversos fatores de virulência de Listeria, a proteína de membrana internalina A (InlA), é umas das mais bem caracterizadas, sendo um excelente alvo por ser altamente exposta na superfície e exclusiva de espécies patogênicas. Neste trabalho é relatado a produção, caracterização e utilização em métodos de diagnósticos de um painel de MAbs contra a InlA (2D12, 3B7, 4E4), e de um MAb (3F8) que reconhece especificamente todas as bactérias do gênero Listeria. Na produção dos MAbs, camundongos BALB/c foram imunizados com uma proteína recombinante InlA (rInlA) concomitantemente com L. monocytogenes inativadas por fervura. Os MAbs gerados demonstraram excelente reatividade por ELISA indireto, Western blot e imunofluorescência. O MAb anti-InlA 2D12 marcado com Cy5 foi usado como anticorpo de detecção de L. monocytogenes, no sistema tipo sanduíche de sensor de fibra óptica. Usando MAb-2D12 como anticorpo de captura nas fibras ópticas, obteve-se um limite de detecção de ~3 x 102 CFU.mL-1, e um limite de detecção de ~1 x 105 CFU.mL-1 foi visualizado com MAb-3F8 como captura. Os MAbs anti-InlA 2D12 e anti-Listeria 3F8 foram posteriormente utilizados para sensibilizar esferas paramagnéticas e testados na separação imunomagnética (IMS) de L. monocytogenes em culturas puras, e em queijo e salsichas tipo hotdog artificialmente contaminados. Após a captura por IMS, as bactérias foram liberadas, incubadas com a fibra óptica ou plaqueadas em agares para contagem. Em paralelo, a confirmação da captura de L. monocytogenes foi realizada por PCR quantitativo em tempo real e por light-scattering technology (BARDOT). Utilizando IMS para separar e concentrar L. monocytogenes, seguido da utilização em plataforma de fibra óptica, foi possível realizar a detecção em menos de 22 horas, de aproximadamente 40 UFC/g de L. monocytogenes em presença de L. innocua, em queijo e salsicha artificialmente contaminados. Além disso, a proteína alvo do MAb3F8 foi identificado como frutose 1,6-bifosfato aldolase através de espectrometria de massa (MALDI-TOF-MS). Os resultados obtidos nesse trabalho indicam que a utilização em conjunto dos sistemas de IMS e fibra óptica com os MAb-2D12 e MAb3F8, foram confiáveis e rápidos, e assim, podendo ser empregados em imunoensaios de rotina para detecção de L. monocytogenes em alimentos. Contudo, ambos MAbs possuem ainda grande potencial para serem mais explorados em outras plataformas de biossensores, assim como, em outros imunoensaios de detecção e funcionalidade de InlA e FBA em Listeria

<p>The crossing of host barriers (intestinal, blood-brain, and placental) is a critical step for systemic infections caused by entero-invasive pathogens. In the intestine, the epithelial cells are the first line of defense against enteric pathogens. <i>Listeria monocytogenes</i> is a facultative-intracellular foodborne pathogen that first crosses the intestinal barrier to cause a systemic infection. However, the underlying mechanism is not well understood.</p><p><br></p> <p>We demonstrate that <i>Listeria</i> adhesion protein (LAP) promotes the translocation of <i>L. monocytogenes </i>across the intestinal barrier in mouse models (A/J and C57BL/6). Relative to the wild-type (WT; serotype 4b) or the isogenic bacterial invasion protein Internalin A mutant (Δ<i>inlA</i>) strain, the <i>lap^─</i> strain showed significant defect in translocation across the intestinal barrier and colonization of the mesenteric-lymph nodes, liver and spleen in the early phase of infection (24 h and 48 h). LAP induces intestinal epithelial barrier dysfunction for increased translocation as evidenced by increased permeability to 4-kDa FITC-dextran (FD4), a marker of paracellular permeability, in the serum and urine of WT and Δ<i>inlA</i>- infected mice and across Caco-2 cell barrier, but not the <i>lap^─</i> mutant strain. Microscopic examination confirmed localization of the WT and Δ<i>inlA</i> strains in the tight junction, a crucial barrier of intestinal paracellular permeability, in the mouse ileal tissue but the <i>lap^─</i> strain remained confined in the lumen. LAP also upregulates TNF-α and IL-6 in intestinal epithelia of mice and in Caco-2 cells for increased permeability. </p><p><br></p> <p>Investigation of the underlying molecular mechanisms of LAP-mediated increase in intestinal permeability by using <i>lap^─</i> mutant strain, purified LAP and shRNA-mediated Hsp60 suppression, we demonstrate that LAP interacts with its host receptor, Hsp60, and activates the canonical NF-κB signaling, which in turn facilitates myosin light-chain kinase (MLCK)-mediated opening of the epithelial barrier via the cellular redistribution of major epithelial junctional proteins claudin-1, occludin, and E-cadherin. Pharmacological inhibition of NF-κB or MLCK in cells or genetic ablation of MLCK in mice (C57BL/6) prevents mislocalization of epithelial junctional proteins, intestinal permeability and <i>L. monocytogenes</i> translocation across the intestinal barrier.</p> <p><br></p><p>Furthermore, LAP also promotes <i>L. monocytogenes </i>translocation across the intestinal barrier and systemic dissemination in a Mongolian gerbil that are permissive to the bacterial invasion proteins; InlA-and InlB-mediated pathways; similar to that in humans. We show a direct LAP-dependent and InlA-independent pathway<i> </i>for <i>L. monocytogenes</i> paracellular translocation across the intestinal epithelial cells that do not express luminally accessible E-cadherin. Additionally, we show a functional InlA/E-cadherin interaction pathway that aids <i>L. monocytogenes</i> translocation by targeting cells with luminally accessible E-cadherin such as cells at the site of epithelial cell extrusion, epithelial folds and mucus-expelling goblet cells. Thus, <i>L. monocytogenes</i> uses LAP to exploit epithelial innate defense in the early phase of infection to cross the intestinal epithelial barrier, independent of other invasion proteins.</p><p><br></p> <p>This work fills a critical gap in our understanding of <i>L. monocytogenes </i>pathogenesis and sheds light to the complex interplay between host-pathogen interactions for bacterial crossing of the crucial intestinal barrier.</p> <br>