Th.--Pharm.--Paris 5, 1984. N°: 97.

Infecções por Listeria monocytogenes têm sido frequentemente relatadas em vários casos de surtos de infecção alimentar pelo mundo. Logo, a preocupação em tornar produtos alimentares de larga escala livres destes patógenos tem aumentado ao longo dos anos. Uma das estratégias mais eficazes são a utilização de bacteriocinas como agentes conservantes, prevenindo a multiplicação bacteriana durante os processos de fabricação, estocagem e distribuição dos produtos. A nisina é uma das bacteriocinas mais conhecidas, sendo empregada na indústria alimentícia por muitos anos. Seu mecanismo de atividade antimicrobiana principal é através da formação de um complexo, juntamente com um precursor de parede celular (lipídio II), que formam poros na membrana celular, causando o extravasamento de conteúdos celulares vitais e a perda de estabilidade eletrolítica, levando à morte celular. Entretanto, algumas evidências apontam para mecanismos alternativos, mas ainda desconhecidos, de morte celular. Uma das abordagens interessantes é por meio da análise dos processos celulares (comparado a uma condição não tratada com nisina) através de metodologias proteômicas. Os cultivos bacterianos foram tratados com concentrações subletais de nisina e os extratos proteicos foram processados em espectrometria de massas em tandem acoplado a um sistema de cromatografia líquida (LC-MS/MS). Os resultados mostraram expressão diferencial de algumas proteínas que atuam contra o estresse oxidativo, como a catalase e proteínas de armazenamento de íons ferrosos. Também verificou-se a superexpressão de uma HSP, a qual pode alterar o dobramento correto de algumas proteínas como a de divisão celular FtsZ. Por fim, a subexpressão de uma chaperona responsável pelo correto dobramento das penicillin binding proteins (PBPs) e a superexpressão de enzimas responsáveis pela síntese de lipídios precursores da membrana celular podem apontar para um sistema de divisão celular alternativo, agindo provavelmente como uma resposta à presença de membranas cobertas por complexos nisina e lipídio II. / Listeria monocytogenes infections have been frequently reported in many food poisoning outbreaks around the world. Therefore, the concern about protecting largely-scale food products from these pathogens has been rising over the years. One of the most efficient strategies is using bacteriocins as a conserving agent, preventing the growth of pathogenic bacteria during the production, storage and distribution of the product. Nisin is a well-known bacteriocin, which has been applied in the food industry for many years. Its main antimicrobial mechanism is based in forming a cell membrane pore creator complex, which coupled with a cell wall precursor (lipid II), leads to the leakage of essential cell life compounds and the loss of electrolytic stability, causing the cell death. However, recent evidences lead to an alternative, but still unknown, cell death mechanism. One interesting approach is analyzing the cell processes (compared to a non-nisin treated condition) by a proteomic approach. The L. monocytogenes cells were treated with a sublethal concentration of nisin and the protein extracts were ran through a tandem mass spectrometry attached to a liquid chromatography system (LC-MS/MS). The results showed differential expression of some agents against oxidative stress such as catalase and ferrous ions storage proteins. Furthermore, it had also presented upregulation of a HSP which can alter the correct folding of other proteins, such as the FtsZ cell division protein. Finally, the downregulation of a chaperone that is responsible of the correct folding of penicillin binding proteins (PBPs) and the superexpression of some enzymes related to the production of cell membrane lipids could point out to a different bacterial cell division system, acting probably as a response to the nisin-lipid II complexes covered cell membranes.

Infecções por Listeria monocytogenes têm sido frequentemente relatadas em vários casos de surtos de infecção alimentar pelo mundo. Logo, a preocupação em tornar produtos alimentares de larga escala livres destes patógenos tem aumentado ao longo dos anos. Uma das estratégias mais eficazes são a utilização de bacteriocinas como agentes conservantes, prevenindo a multiplicação bacteriana durante os processos de fabricação, estocagem e distribuição dos produtos. A nisina é uma das bacteriocinas mais conhecidas, sendo empregada na indústria alimentícia por muitos anos. Seu mecanismo de atividade antimicrobiana principal é através da formação de um complexo, juntamente com um precursor de parede celular (lipídio II), que formam poros na membrana celular, causando o extravasamento de conteúdos celulares vitais e a perda de estabilidade eletrolítica, levando à morte celular. Entretanto, algumas evidências apontam para mecanismos alternativos, mas ainda desconhecidos, de morte celular. Uma das abordagens interessantes é por meio da análise dos processos celulares (comparado a uma condição não tratada com nisina) através de metodologias proteômicas. Os cultivos bacterianos foram tratados com concentrações subletais de nisina e os extratos proteicos foram processados em espectrometria de massas em tandem acoplado a um sistema de cromatografia líquida (LC-MS/MS). Os resultados mostraram expressão diferencial de algumas proteínas que atuam contra o estresse oxidativo, como a catalase e proteínas de armazenamento de íons ferrosos. Também verificou-se a superexpressão de uma HSP, a qual pode alterar o dobramento correto de algumas proteínas como a de divisão celular FtsZ. Por fim, a subexpressão de uma chaperona responsável pelo correto dobramento das penicillin binding proteins (PBPs) e a superexpressão de enzimas responsáveis pela síntese de lipídios precursores da membrana celular podem apontar para um sistema de divisão celular alternativo, agindo provavelmente como uma resposta à presença de membranas cobertas por complexos nisina e lipídio II. / Listeria monocytogenes infections have been frequently reported in many food poisoning outbreaks around the world. Therefore, the concern about protecting largely-scale food products from these pathogens has been rising over the years. One of the most efficient strategies is using bacteriocins as a conserving agent, preventing the growth of pathogenic bacteria during the production, storage and distribution of the product. Nisin is a well-known bacteriocin, which has been applied in the food industry for many years. Its main antimicrobial mechanism is based in forming a cell membrane pore creator complex, which coupled with a cell wall precursor (lipid II), leads to the leakage of essential cell life compounds and the loss of electrolytic stability, causing the cell death. However, recent evidences lead to an alternative, but still unknown, cell death mechanism. One interesting approach is analyzing the cell processes (compared to a non-nisin treated condition) by a proteomic approach. The L. monocytogenes cells were treated with a sublethal concentration of nisin and the protein extracts were ran through a tandem mass spectrometry attached to a liquid chromatography system (LC-MS/MS). The results showed differential expression of some agents against oxidative stress such as catalase and ferrous ions storage proteins. Furthermore, it had also presented upregulation of a HSP which can alter the correct folding of other proteins, such as the FtsZ cell division protein. Finally, the downregulation of a chaperone that is responsible of the correct folding of penicillin binding proteins (PBPs) and the superexpression of some enzymes related to the production of cell membrane lipids could point out to a different bacterial cell division system, acting probably as a response to the nisin-lipid II complexes covered cell membranes.

Infecções por Listeria monocytogenes têm sido frequentemente relatadas em vários casos de surtos de infecção alimentar pelo mundo. Logo, a preocupação em tornar produtos alimentares de larga escala livres destes patógenos tem aumentado ao longo dos anos. Uma das estratégias mais eficazes são a utilização de bacteriocinas como agentes conservantes, prevenindo a multiplicação bacteriana durante os processos de fabricação, estocagem e distribuição dos produtos. A nisina é uma das bacteriocinas mais conhecidas, sendo empregada na indústria alimentícia por muitos anos. Seu mecanismo de atividade antimicrobiana principal é através da formação de um complexo, juntamente com um precursor de parede celular (lipídio II), que formam poros na membrana celular, causando o extravasamento de conteúdos celulares vitais e a perda de estabilidade eletrolítica, levando à morte celular. Entretanto, algumas evidências apontam para mecanismos alternativos, mas ainda desconhecidos, de morte celular. Uma das abordagens interessantes é por meio da análise dos processos celulares (comparado a uma condição não tratada com nisina) através de metodologias proteômicas. Os cultivos bacterianos foram tratados com concentrações subletais de nisina e os extratos proteicos foram processados em espectrometria de massas em tandem acoplado a um sistema de cromatografia líquida (LC-MS/MS). Os resultados mostraram expressão diferencial de algumas proteínas que atuam contra o estresse oxidativo, como a catalase e proteínas de armazenamento de íons ferrosos. Também verificou-se a superexpressão de uma HSP, a qual pode alterar o dobramento correto de algumas proteínas como a de divisão celular FtsZ. Por fim, a subexpressão de uma chaperona responsável pelo correto dobramento das penicillin binding proteins (PBPs) e a superexpressão de enzimas responsáveis pela síntese de lipídios precursores da membrana celular podem apontar para um sistema de divisão celular alternativo, agindo provavelmente como uma resposta à presença de membranas cobertas por complexos nisina e lipídio II. / Listeria monocytogenes infections have been frequently reported in many food poisoning outbreaks around the world. Therefore, the concern about protecting largely-scale food products from these pathogens has been rising over the years. One of the most efficient strategies is using bacteriocins as a conserving agent, preventing the growth of pathogenic bacteria during the production, storage and distribution of the product. Nisin is a well-known bacteriocin, which has been applied in the food industry for many years. Its main antimicrobial mechanism is based in forming a cell membrane pore creator complex, which coupled with a cell wall precursor (lipid II), leads to the leakage of essential cell life compounds and the loss of electrolytic stability, causing the cell death. However, recent evidences lead to an alternative, but still unknown, cell death mechanism. One interesting approach is analyzing the cell processes (compared to a non-nisin treated condition) by a proteomic approach. The L. monocytogenes cells were treated with a sublethal concentration of nisin and the protein extracts were ran through a tandem mass spectrometry attached to a liquid chromatography system (LC-MS/MS). The results showed differential expression of some agents against oxidative stress such as catalase and ferrous ions storage proteins. Furthermore, it had also presented upregulation of a HSP which can alter the correct folding of other proteins, such as the FtsZ cell division protein. Finally, the downregulation of a chaperone that is responsible of the correct folding of penicillin binding proteins (PBPs) and the superexpression of some enzymes related to the production of cell membrane lipids could point out to a different bacterial cell division system, acting probably as a response to the nisin-lipid II complexes covered cell membranes.

Orientador: Mary Luci de Souza Queiroz / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-04T00:31:06Z (GMT). No. of bitstreams: 1 Resta_AndreiadosSantos_M.pdf: 837966 bytes, checksum: ca84712f5cd6dd4ba54f60e463697d96 (MD5) Previous issue date: 2004 / Resumo: Neste trabalho foi investigado o efeito imunomodulador do extrato seco de Spirulina sp sobre o crescimento e diferenciação de precursores hematopoéticos de granulócitos-macrófagos (CFU-GM) na medula óssea e no baço de camundongos BALB/c infectados com Listeria monocytogenes. Alterações no peso do baço e na resistência dos animais à infecção também foram estudadas. Foram testadas quatro doses do extrato de Spirulina sp: 50, 150, 200 e 300 mg/kg/dia, administradas por via oral aos animais. Três protocolos de tratamento foram utilizados para avaliar os efeitos da alga sobre a resistência de camundongos infectados intraperitonealmente com uma dose letal Listeria monocytogenes (6x104 bactérias/animal). No primeiro protocolo, animais infectados foram pré-tratados por 7 dias com as diferentes doses do extrato. No segundo, doses de 150 e 200 mg/kg/dia foram administradas aos animais por 14 dias consecutivos, sendo que a suspensão de bactérias foi inoculada no 7° dia de tratamento. No terceiro protocolo, os animais foram submetidos a um pós-tratamento de 7 dias com essas mesmas doses de extrato. Para avaliação dos parâmetros hematopoéticos foi utilizado apenas o protocolo de pré-tratamento e os animais foram sacrificados 24, 48 e 72 h após inoculação intraperitoneal de uma dose subletal de Listeria monocytogenes (1x105 bactérias/animal). Animais infectados com uma dose subletal de Listeria monocytogenes apresentaram um decréscimo significativo no número de CFU-GM da medula óssea 48 e 72 h após a infecção. Esse efeito foi acompanhado por um aumento no número dessas células no baço assim como no peso deste órgão. Todas as doses de Spirulina utilizadas protegeram contra a mielossupressão provocada pela bactéria, porém um aumento estatisticamente significativo neste parâmetro foi obtido para as doses de 150 e 200 mg/kg/dia em relação ao controle e às outras doses. Estimulação da mielopoese também foi observada nos grupos de animais normais (não infectados) tratados por 7 dias com 150 e 200 mg/kg/dia de Spirulina em relação aos outros grupos. Além disso, o pré-tratamento dos animais infectados com todas as doses avaliadas inibiu o desenvolvimento da esplenomegalia e da hematopoese esplênica. Nenhuma alteração foi observada no baço dos animais apenas tratados. Empregando-se esse mesmo protocolo de pré-tratamento, as doses de 150 e 200 mg/kg/dia também aumentaram a resistência de camundongos letalmente infectados com Listeria monocytogenes, concordando com os resultados obtidos na avaliação dos parâmetros hematopoéticos. Quando o tratamento foi prolongado para 14 dias com essas mesmas doses de extrato e os animais infectados no 7o dia de tratamento, observou-se um aumento estatisticamente significativo de 35% e 30% na probabilidade de sobrevida dos animais infectados que receberam 150 e 200 mg/kg/dia da alga, respectivamente. No entanto, nenhuma alteração no tempo de sobrevida de animais infectados foi observada com o protocolo de pós-tratamento por 7 dias com 150 e 200 mg/kg/dia de extrato de Spirulina sp. Estes resultados apontam para um efeito imunoestimulante da alga quando utilizada profilaticamente e sugerem que o aumento na resistência do hospedeiro à infecção depende, em parte, do protocolo utilizado. Neste sentido, a administração do extrato seco de Spirulina sp previamente à infecção parece ser fundamental para aumentar a resistência imunológica do hospedeiro, provavelmente devido à estimulação da geração de precursores hematopoéticos de granulócitos e macrófagos, críticos para a defesa inicial do organismo contra a infecção bacteriana / Abstract: In this work, we investigated the effects of Spirulina sp extract on the growth and differentiation of bone marrow and spleen hematopoietic progenitors (CFU-GM) in normal and in Listeria monocytogenes-infected mice. Changes in spleen weight and resistance to a lethal dose of bacteria were also studied. To evaluate the hematopoietic activity, BALB/c mice were treated orally with 50, 150, 200 and 300 mg/kg doses of the extract for 7 consecutive days and, at the end of this period, they were infected intraperitoneally with a sublethal dose of the bacteria (1x103 bacteria/animal). As expected, a significant decrease in bone marrow CFU-GM numbers was observed in mice infected with L. monocytogenes at 48 and 72 h after infection. This effect was accompanied by the development of splenic hematopoiesis with splenomegaly. Pre-treatment of these animals with Spirulina sp significantly stimulated myelopoiesis, reaching normal values of bone marrow CFU-GM when 50 and 300 mg/kg of the algae were used. On the other hand, increased numbers of bone marrow CFU-GM over control values were observed when the extract was given to mice at 150 and 200 mg/kg previously to infection. Moreover, these doses also stimulated myelopoiesis in normal mice given the extract for 7 days. All of these doses of Spirulina sp completely inhibited the extramedullar hematopoiesis and the increase in spleen weight induced by the infection. This extract did not affect splenic hematopoiesis and spleen weight when administered to normal mice. Resistance to infection was studied in mice infected with a lethal dose of L. monocytogenes (6x104 bacteria/animal) and submitted to 3 protocols of treatment with Spirulina sp. These experiments show that only the doses of 150 and 200 mg/kg given for 7 days to mice previously to infection were effective to prolong survival of these animals until 12 days, compared with non-treated infected mice which died until 6 days. When 150 and 200 mg/kg of the extract were administered to mice for 14 consecutive days and the animals were infected at the 7th day of treatment, 30 and 35% of survival were observed, respectively. In contrast, post-treatment of infected mice with these doses did not affect survival, suggesting an important role for the pre-treatment with Spirulina sp in the prophylaxis of bacterial infections. Taken together, these results suggest that the stimulatory effect of Spirulina sp on myelopoiesis is critically important to improve resistance of L. monocytogenes-infected mice. Moreover, the present results support previous work in the literature suggesting the innate immune system as a major target of Spirulina-mediated immune activation / Mestrado / Mestre em Farmacologia

Philosophiae Doctor - PhD / Listeria monocytogenes has been established as a food-borne pathogen since the early 1980s and has become a big concern for the food industry and Public Health authorities (Doyle 2001; Oliveira et al. 2003; Capita et al. 2005; Conly and Johnston 2008). It is a Gram-positive, opportunistic facultative intracellular bacterium which is frequently present in nature and may be found in any food environment (Liu 2006; Chen et al. 2007; Conly and Johnston 2008). Of the six species of Listeria, L. monocytogenes is the only one capable of causing listeriosis, a severe food-borne illness in humans (de Vasconcelos et al. 2008). For the average healthy person, although the incidence of infection is low, symptoms of febrile gastroenteritis may be presented (Gianfranceschi et al. 2007; Kersting et al. 2010). In immunocompromised individuals however, the hospitalization and mortality rates are amongst the highest for pathogenic organisms (Tran and Kathariou 2002; Lin et al. 2006; Schuppler and Loessner 2010). Illnesses such as septicaemia and central nervous system infections may also occur in these individuals (Roberts et al. 2006; Schuppler and Loessner 2010). Pregnant women and their fetus are also largely at risk where pre-term delivery and birth defects may occur as a result of listeriosis (Doyle 2001; Garrido et al. 2008). The epidemiological surveillance systems for the reporting of listeriosis are poor as it is a non-notifiable disease in many countries. Therefore, the incidence of infection that is regarded as low must be reconsidered (Mammina et al. 2009; Pinto et al. 2010). Listeria monocytogenes can reproduce in a wide variety of reservoirs within food processing plants, thereby contaminating the food which then poses a risk for food-borne illness. It can be transmitted from infected animals to humans and also through the consumption of foods from animal origin (Kalender 2003; Kersting et al. 2010). Animals are infected by Listeria spp. found in the environment; the organism is then transmitted through the blood, milk and excrement of the animal back into the environment where manure, soil, feed and water can become contaminated again (Akpolat et al. 2004; Kersting et al. 2010). Poultry products and ready-to-eat (RTE) food that support the growth of L. monocytogenes, including soft cheeses, unpasteurized milk, hotdogs, deli meats, vegetables and fruits have been linked to cases of listeriosis (Rørvik et al. 2003; Chen et al. 2007; Conly and Johnston 2008; Ford 2010; Kersting et al. 2010). Regardless of HACCP systems that are in place in the food processing plants, listeriosis outbreaks still occur as a result of the ingestion of these food products. Serotyping, based on the serological reaction between somatic (O) and flagellar (H) antigens and their corresponding sera, has identified 13 L. monocytogenes serotypes (Nadon et al. 2001; Wiedmann 2002; Kérouanton et al. 2010). Of the 13 serotypes of L. monocytogenes, 1/2a, 1/2b and 4b are responsible for more than 95% of listeriosis infections in humans (Mereghetti et al. 2002; Moorhead et al. 2003; Borucki et al. 2004;de Vasconcelos et al. 2008). L. monocytogenes serotypes 1/2a and 1/2b are mainly associated and isolated sporadically from food and 4b is responsible for the major human epidemic cases (Gilbreth et al. 2005). L. monocytogenes serotypes 1/2a and 1/2b are also responsible for sporadic cases of human illness (Wiedmann 2002). / South Africa

Listeria monocytogenes causes a serious foodborne illness (listeriosis) with a fatality rate of about 30% in susceptible individuals (1). Timely identification of foods and food processing environments carrying this deadly bacterium is crucial for implementing effective interventions but remains a practical challenge due to the complexity of test samples, low level of bacterial contamination, and the ubiquity and the genetic diversity of Listeria isolates. The purpose of this work was to identify and assess surface proteins of L. monocytogenes that can serve as diagnostic biomarkers for pathogen isolation and detection using antibody-based methods. Bioinformatics analysis of 130 putative surface proteins encoded by the genome of L. monocytogenes F2365 (serotype 4b) revealed four uncharacterized proteins with extensive amino acid sequences unique to L. monocytogenes. These proteins did not contain identifiable PrfA-controlled promoter elements. The four proteins were expressed at the transcriptional level in vitro, as demonstrated by RT-PCR, but only one of the four proteins, LMOf2365_0639, was detected on the cell surface by immunofluorescence microscopy (IFM) using rabbit polyclonal antibodies (PAbs) raised against corresponding recombinant proteins. Transcription start site mapping and promoter prediction analysis provided evidence that the LMOf2365_0639 gene was expressed under the control of a sigma B factor-dependent promoter, an alternative sigma factor involved in stress response. Non-gel based proteomics analysis of L. monocytogenes surface proteins identified 36 surface proteins in at least one of the three trials performed. IFM with PAbs raised against each of the five candidate surface proteins identified from the proteomics study revealed a strong fluorescence signal on the surface of live L. monocytogenes cells with LMOf2365_0148 specific PAbs, indicating a good level of expression of this protein. These results suggested the potential of the surface proteins LMOf2365_0639 and LMOf2365_0148 as diagnostic biomarkers for L. monocytogenes. Thirty-five and 24 monoclonal antibodies (MAbs) were developed against purified recombinant LMOf2365_0639 and LMOf2365_0148, respectively. Three MAbs against LMOf2365_0639 and five MAbs against LMOf2365_0148 were selected and evaluated for their potential in L. monocytogenes detection and isolation based on the observation that these MAbs recognized the highest number of the 53 L. monocytogenes isolates and the lowest number of the 10 other Listeria species isolates tested. None of these MAbs reacted with the four foodborne pathogens (Campylobacter jejuni, Samonella enterica serovar Typhimurium, Escherichia coli O157:H7 and Bacillus cereus) tested. All three MAbs to LMOf2365_0639 were specific for lineage I and II isolates of L. monocytogenes commonly found in clinical and food isolates respectively and recognized the N-terminal region of LMOf2365_0639. Anti-LMOf2365_0148 MAbs were reactive to lineage I and lineages III L. monocytogenes isolates commonly found in clinical and animal isolates respectively. Both LMOf2365_0639 and LMOf2365_0148 were expressed in standard enrichment culture conditions according to Health Canada’s MFHPB-30 and MFHPB-07 methods. In addition, MAbs against LMOf2365_0148 could specifically isolate live L. monocytogenes by immunomagnetic separation even in a mixture of L. monocytogenes and non-target L. innocua. The dissociation constants of the MAbs capable of capturing L. monocytogenes ranged from 2.58 x 10-8 M to 8.87 x 10-10 M. In conclusion, two novel surface proteins LMOf2365_0639 and LMOf2365_0148 were identified, were shown to be expressed in L. monocytogenes grown in standard selective enrichment cultures, and can be explored as surface biomarkers for the isolation and detection of L. monocytogenes with specific MAbs developed in this study.

Orientador: Tomomasa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-03T15:36:39Z (GMT). No. of bitstreams: 1 Marques_EneidaGoncalvesLemes_M.pdf: 6403857 bytes, checksum: aba606a1028881cdd6692f20d1eb780b (MD5) Previous issue date: 2003 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic digital document / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Cholesterol has been shown to play important role in the pathogenesis and persistence of intracellular pathogens. Here, we modulate host cholesterol biosynthesis pathway using pharmacological agent statins, which are reversible inhibitors of HMG†CoA reductase enzyme. The aim of the study was to investigate the role of statins in inducing host protective responses against intracellular pathogens. We report reduced growth of Listeria monocytogenes (LM) and Mycobacterium tuberculosis (Mtb) in murine macrophages. We show prominent immunomodulatory activity induced by statins, mainly increased phagosomal maturation and autophagy resulting in decreased bacterial growth in macrophages. Subsequently, statin†treated mice showed decrease in bacterial loads, accompanied by reduced histopathology in the acute phase of infection during listeriosis and tuberculosis. Furthermore, we found decreased growth of Mtb in peripheral blood mononuclear cells (PBMC) and monocyte†derived macrophages (MDM) isolated from patients with familial hypercholesterolemia (FH) on statin therapy when compared to healthy subjects. Together, our results show that statins induces protection against Mtb in murine macrophages, mice and human mononuclear cells and monocyte†derived macrophages.

The pathogenicity of food-borne intracellular bacterium Listeria monocytogenes is greatly associated with its abilities to invade non-phagocytic cells, counteract the host innate immune system, resist bactericidal antibiotic-mediated killing, and breaking the physical barriers. In the last 30 years of research on L. monocytogenes, several virulence factors, such as Listeriolysin O (LLO), InlA, InlB, ActA, PI-PLC, and PC-PLC have already been characterized as important players that help this bacterium to achieve the key stage of infection. There are approximately 3,000 open reading frames in Listeria’s genome; however, only few virulence factors are functionally characterized. Thus, it is important to identify new virulence factors and understand how new virulence factors in Listeria help this opportunistic pathogen to counteract the host innate immune system, resist antibiotic-mediated killing, colonize vital organs, and finally successfully develop life-threatening listeriosis. In this study, inrame deletion mutagenesis was used to generate the deletion mutants of novel listerial virulence factors and a series of biochemical, in vitro and in vivo experiments were conducted to characterize the roles of these virulence factors during the infection process. In the first part of this study, an AlkD-like protein (Adlp, LmoF2365_0220) was identified and the protein is associated with oxidant tolerance and aminoglycoside antibiotic resistance. In the second part of this study, a new internalin-like protein (LmoH7858_0369) was shown to be involved in invasion of Hep-G2 cells and organ colonization in mice. The third part of this study showed that listeriolysin O (LLO) mediates cytotoxicity on brain endothelial cells, suggesting that LLO may contribute to the invasion of the central nervous system by L. monocytogenes. In summary, we identified and characterized two novel virulence factors, Adlp and LmoH7858_0369 that contributed to bacterial infection and revealed a new invasion mechanism of CNS cells that is mediated by LLO. Results from these studies provide a better understanding on the pathogenicity of L. monocytogenes and can be used as therapeutical targets or vaccine candidates