Aurora M. Cianciarullo

Production of Human Papillomavirus Type 16 L1 Virus-Like Particles by Recombinant Lactobacillus casei Cells

ABSTRACT Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on the Lactobacillus casei lactose operon promoter (plac) for expression of the HPV-16 L1 protein in L. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis of L. casei expressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on the L. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized with L. casei expressing L1 reacted with Spodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs by Lactobacillus opens the possibility for development of new live mucosal prophylactic vaccines.

Functional characterization of LcpA, a surface-exposed protein of Leptospira spp. that binds the human complement regulator C4BP.

ABSTRACT We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface‐exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection.

Differentiation of typical and atypical enteropathogenic Escherichia coli using colony immunoblot for detection of bundle-forming pilus expression

Aims:  The aim of study was to develop a colony immunoblot assay to differentiate typical from atypical enteropathogenic Escherichia coli (EPEC) by detection of bundle‐forming pilus (BFP) expression.

Comparison of the pulmonary response against lethal and non-lethal intranasal challenges with two different pneumococcal strains

The differences between the immune response elicited during a self-limiting and a life-threatening lung infection with Streptococcus pneumoniae was analyzed in a mouse model of intranasal challenge using two different pneumococcal strains. M10, a serotype 11A strain, induced an early response within the first 12h after the challenge, which was characterized by the early local secretion of TNF-alpha and IL-6, followed by a sharp and rapid neutrophil influx. Bacterial loads in the lungs already started to fall at 12h after the challenge and no pneumococci could be recovered after 36h, at the time point when the animals started to show improvement in disease symptoms. ATCC6303, a serotype 3 strain, on the other hand, showed only a late increase in local TNF-alpha and IL-6 levels, when bacterial growth already seems to be out of control. Although cell influx was also observed, neutrophil rise was not as marked as with M10 (type 11A). Pneumococcal loads increased constantly and bacteria started to be recovered from the blood at 30h after the challenge. After this time point, animals showed worsening of symptoms and became lethargic. The resolution of the acute infection could be thus correlated with the early induction of proinflammatory cytokines, which could be due to the presence of a thinner polysaccharide capsule in M10 (type 11A), rendering bacterial components capable of activating the innate immune response more accessible.

A group IIA‐secreted phospholipase A2 from snake venom induces lipid body formation in macrophages: the roles of intracellular phospholipases A2 and distinct signaling pathways

We investigated the ability of the sPLA2, known as MT‐III, isolated from the viperid snake Bothrops asper, to induce LB formation in macrophages and the major cellular signaling pathways involved in this process. The effects of MT‐III on ADRP localization and expression and macrophage ultrastructure were assessed. Our results showed that this sPLA2 induced a marked increase in LB numbers in macrophages, induced the recruitment of ADRP in macrophages, and up‐regulated ADRP expression. Ultrastructural analysis showed the presence of weakly and strongly osmiophilic LBs in sPLA2‐stimulated cells. Enlargement of the ER and Golgi cisterns was also observed. Pretreatment of cells with H7 or staurosporine (PKC inhibitors), LY294002 or wortmannin (PI3K inhibitors), SB202190 or PD98059 (p38MAPK and ERK1/2 inhibitors, respectively), or Pyr‐2 or Bel (cPLA2 and iPLA2 inhibitors, respectively) significantly reduced sPLA2‐induced LB formation. Herbimycin (a PTK inhibitor) and indomethacin or etoricoxib (COX inhibitors) failed to alter sPLA2‐induced effects. In conclusion, our results show for the first time the ability of a venom sPLA2 to induce the formation of LBs and the expression of ADRP in macrophages. Venom PLA2‐induced LB formation is dependent on PKC, PI3K, p38MAPK, ERK1/2, cPLA2, and iPLA2 signaling pathways but not on PTK, COX‐1, or COX‐2 pathways. Activation of the ER and Golgi complex may play an important role in the formation of LBs induced by this sPLA2 in macrophages.

Investigation of influenza in migrating birds, the primordial reservoir and transmitters of influenza in Brazil

Birds are the most important reservoirs of the influenza virus. Its maintenance in its natural hosts, including man, allows the influenza virus to reassorts its strains. The recent report of an avian influenza A (H5N1) virus in humans, was in a child with fatal respiratory illness in China, 1997. The current study was conducted to elucidate the transportation of the influenza by birds that migrate, annually, through the both Northern and Southern hemispheres, with special attention paid to the Vireo olivaceus [Juruviara(BR) or Red-eyed vireo(USA)] species, which travels from the USA to Brazil, and vice versa, and the Elaenia mesoleuca [Tuque(BR) or (USA)] species that flies over the entire Southern Hemisphere. There are two species of birds, which breed and migrate in Sao Paulo State, Brazil, and which were demonstrated to carry Influenza virus, were selected. The viral particles isolated were observed by electron microscopy. The influenza virus was detected by the House Duplex/PCR and Gloria molecular biology tests. The results demonstrated that the Elaenia mesoleuca and Vireo olivaceus bird species are carrying the Influenza virus whilst crossing both the Northern and Southern hemispheres. To understand the role that these migrating birds may play in epidemic influenza, in Brazil, characterization of avian influenza subtypes will be done.

Influenza em animais heterotérmicos

The objective was to study Orthomyxovirus in heterothermic animals. Blood samples from snakes (genus Bothrops and Crotalus) and from toads and frogs (genus Bufo and Rana) were collected to evaluate the red cell receptors and antibodies specific to influenza virus by the hemagglutination and hemagglutination inhibition tests, respectively. Both snakes and toads kept in captivity presented receptors in their red cells and antibodies specific to either influenza virus type A (human and equine origin) or influenza type B. The same was observed with recently captured snakes. Concerning the influenza hemagglutination inhibition antibodies protective levels were observed in the reptiles’ serum, against influenza type A and type B. Unlike the toads, 83.3% of the frogs presented mean levels of Ab 40HIU for some influenza strains. It was concluded that heterothermic animals could offer host conditions to the influenza virus and also susceptibility to the infection. Key-words: Orthomyxovirus. Heterothermic animals. Receptors. Specific antibodies. Influenza virus. 1. Laboratorio de Virologia do Instituto Butantan, Sao Paulo, SP. 2. Laboratorio de Genetica do Instituto Butantan, Sao Paulo, SP. 3. Laboratorio de Herpetologia do Instituto Butantan, Sao Paulo, SP. Endereco para correspondencia: Dra. Dalva Assuncao Portari Mancini. Divisao de Desenvolvimento Cientifico/Laboratorio de Virologia/IB. Av. Vital Brasil 1500, 05503-900 Sao Paulo, SP. Tel: 55 11 3726-7222 ramal 2152 e-mail: labvirol@bol.com.br dapmancini@butantan.gov.br. Recebido para publicacao em 8/2/2003 Aceito em 27/2/2004 Para melhor entendimento sobre a funcao do receptor celular no reconhecimento do virus influenza, tem sido desenvolvidas pesquisas relacionadas a especificidade de ligacao desse virus isolado de varias especies animais 10 . Bossart e cols verificaram atraves da microscopia eletronica, os fenomenos conhecidos de adsorcao (atraves da hemaglutinina) e subsequente eluicao (pela neuraminidase) do virus influenza sobre a membrana de eritrocitos de ave e de humano. Essa interacao do virus a celula vermelha e comparado ao que ocorre as celulas hospedeiras. Os virus influenza tipo A mostram habilidade, entre seus subtipos, de ligacao aos receptores atraves dos terminais dos acidos sialicos, contendo oligossacarideos, mas, sao distintos quanto a especificidade de receptor, o que provavelmente os diferencia quanto a virulencia aos hospedeiros . Quanto ao tropismo desse virus, verifica-se que as celulas receptoras virus ARTIGO/ARTICLEO objetivo foi pesquisar Ortomyxovirus em animais heterotermicos. Coletou-se sangue de serpentes dos generos Bothrops e Crotalus e de sapo e ras dos generos Bufo e Rana, para a deteccao dos receptores de hemacias e anticorpos especificos, ao virus influenza, pelos testes de hemaglutinacao e inibicao da hemaglutinacao, respectivamente. Pelo teste de hemaglutinacao, verificou-se que serpentes e sapos em cativeiro apresentaram receptores em suas hemacias para o virus influenza, humano e equino do tipo A e tipo B. O mesmo ocorreu com serpentes recem chegadas. Quanto ao teste de inibicao da hemaglutinacao dos soros dos repteis observou-se titulos protetores de anticorpos aos virus influenza tipo A (origens humana e equina) e tipo B. Com soro de sapo nao se observou reacao de inibicao da hemaglutinacao porem, 83,3% das ras obtiveram medias de 40UIH para algumas cepas. Conclui-se que animais heterotermicos podem oferecer condicoes de hospedeiros aos virus influenza, assim como susceptibilidade a infeccao.

Late Establishment of the Attaching and Effacing Lesion Caused by Atypical Enteropathogenic Escherichia coli Depends on Protein Expression Regulated by Per

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is classified as typical (tEPEC) or atypical (aEPEC) based on the presence or absence of the E. coli adherence factor plasmid (pEAF), respectively. The hallmark of EPEC infection is the formation of the attaching and effacing (A/E) lesions on the gut mucosa. We compared the kinetics of A/E lesion formation induced by aEPEC and tEPEC. The examination of infected HEp-2 cells clearly demonstrated delayed A/E lesion formation by aEPEC in comparison to tEPEC. This delay was associated with the expression of locus of enterocyte effacement (LEE)-encoded virulence factors (i.e., intimin and EspD). Indeed, the insertion of a plasmid containing perABC, a transcriptional regulator of virulence factors involved in A/E formation, into aEPEC strains increased and accelerated the formation of A/E lesions. Interestingly, the enhanced expression and translocation of LEE-encoded proteins, such as those expressed in LEE5 (intimin) and LEE4 (EspD), in aEPEC (perABC) was independent of bacterial adhesion. The secretion kinetics of these two proteins representing LEE5 and LEE4 expression correlated with A/E lesion formation. We conclude that the lack of Per in the regulation network of virulence genes is one of the main factors that delay the establishment of A/E lesions induced by aEPEC strains.

Adherence and invasion of Bacteroidales isolated from the human intestinal tract

Members of the genera Bacteroides and Parabacteroides are important constituents of both human and animal intestinal microbiota, and are significant facultative pathogens. In this study, the ability of Bacteroides spp. and Parabacteroides distasonis isolated from both diarrhoeal and normal stools (n = 114) to adhere to and invade HEp-2 cells was evaluated. The presence of putative virulence factors such as capsule and fimbriae was also investigated. Adherence to HEp-2 cells was observed in 75.4% of the strains, which displayed non-localized clusters. Invasion was observed in 37.5% and 26% of the strains isolated from diarrhoeal and non-diarrhoeal stools, respectively. All strains displayed a capsule, whereas none of them showed fimbriae-like structures. This is the first report of the ability of Bacteroides spp. and P. distasonis to adhere to and invade cultured HEp-2 epithelial cells.