No / Streptococcus agalactiae is a major neonatal pathogen. Disease progression is characterised by bacterial adaptation from commensal maternal vaginal colonisation to environments associated with neonatal disease, including exposure to blood. To explore this adaptation in vitro, we have used proteomics to identify proteins differentially expressed following growth on Todd Hewitt agar in the presence or absence of 10% v/v human serum. Twelve differentially expressed proteins were identified. Notably, the C protein β antigen and arginine deiminase proteins were upregulated following growth in the presence of human serum, consistent with previous studies implicating these two proteins in the pathogenesis of S. agalactiae disease.

No / Streptococcus agalactiae is a major neonatal pathogen that is able to adapt to a variety of host environments, including both rectal and vaginal maternal carriage, growth in amniotic fluid and at various neonatal body sites. As such it is important to elucidate the patterns of protein expression that are associated with S. agalactiae growth under these different in vivo conditions. To this end, we have grown S. agalactiae strain A909 under in vitro conditions reflecting those associated with maternal vaginal carriage (low pH, low oxygen, nutrient stress) and those associated with exposure to body fluids during invasive disease (neutral pH, aeration, nutrient sufficient). The protein profiles of bacterial cells grown under each of these conditions were compared using a proteome approach. A total of 76 proteins were reproducibly identified 16 of which were shown to be differentially expressed. The putative virulence factor C protein β and several proteins linked to resistance to oxidative stress were found to be upregulated under the conditions hypothesised to reflect those associated with foetal exposure to S. agalactiae. Thus, these data add to the currently limited understanding of the molecular basis of S. agalactiae GBS adaptation to different environmental conditions.

Yes / Background: Group B Streptococcus (GBS) is a leading cause of morbidity and mortality in infants in both developed and developing countries. To our knowledge, only a few studies have been reported the clinical features, treatment and outcomes of the GBS disease in China. The severity of neonatal GBS disease in China remains unclear. Population-based surveillance in China is therefore required. Methods: We retrospectively collected data of <3 months old infants with culture-positive GBS in sterile samples from three large urban tertiary hospitals in South China from Jan 2011 to Dec 2014. The GBS isolates and their antibiotic susceptibility were routinely identified in clinical laboratories in participating hospitals. Serotyping and multi-locus sequence typing (MLST) were also conducted for further analysis of the neonatal GBS disease. Results: Total 70 cases of culture-confirmed invasive GBS infection were identified from 127,206 live births born in studying hospitals, giving an overall incidence of 0.55 per 1000 live births (95% confidence interval [CI] 0.44–0.69). They consisted of 49 with early-onset disease (EOD, 0.39 per 1000 live births (95% CI 0.29–0.51)) and 21 with late-onset disease (LOD, 0.17 per 1000 live births (95% CI 0.11–0.25)). The incidence of EOD increased significantly over the studying period. Five infants (4 EOD and 1 LOD) died before discharge giving a mortality rate of 7.1% and five infants (7.1%, 2 EOD and 3 LOD) had neurological sequelae. Within 68 GBS isolates from GBS cases who born in the studying hospitals or elsewhere, serotype III accounted for 77.9%, followed by Ib (14.7%), V (4.4%), and Ia (2.9%). MLST analysis revealed the presence of 13 different sequence types among the 68 GBS isolates and ST-17 was the most frequent sequence type (63.2%). All isolates were susceptible to penicillin, ceftriaxone, vancomycin and linezolid, while 57.4% and 51.5% were resistant to erythromycin and clindamycin, respectively. Conclusions: This study gains the insight into the spectrum of GBS infection in south China which will facilitate the development of the guidance for reasonable antibiotics usage and will provide evidence for the implementation of potential GBS vaccines in the future. / Supported by medical and health science and technology projects of Health and Family Planning Commission of Guangzhou Municipality (grant number 20151A010034) and Guangdong provincial science and technology planning projects (grant number 2014A020212520).

Tyrimo tikslas – nustatyti nėščių moterų B grupės β hemolizinio streptokoko (BGS) ir Escherichia coli (E.coli) nešiojimo bei naujagimių kolonizacijos dažnumą ir įvertinti šių mikroorganizmų įtaką naujagimių ankstyvai infekcijai atsirasti. Metodika. Perspektyvinis momentinis stebėjimo tyrimas vykdytas Kauno medicinos universiteto Akušerijos ir ginekologijos bei Neonatologijos klinikose. Moterims du atskiri pasėliai iš makšties apatinio trečdalio ir išangės buvo paimti 35-37 nėštumo savaitę arba gimdymo metu, o naujagimiams - iš ausies išorinės landos bei nosiaryklės per 5 – 15 min. po gimimo. Išskirtų BGS serotipavimas atliktas naudojant 9 specifinius antiserumus, o jautrumas antibiotikams nustatytas diskų difuzijos metodu pagal klinikinių laboratorijų standartus nustatančio komiteto (NCCLS) rekomendacijas. Rezultatai. Šimtas keturiasdešimt aštuonioms moterims iš 970 (15,3 proc.) buvo nustatytas BGS nešiojimas, o 193 moterims (19,9 proc.) - E.coli nešiojimas. Naujagimių BGS ir E.coli kolonizacijos dažnumas buvo 6,4 proc. ir 14,4 proc., o vertikalaus pernešimo – atitinkamai 28,4 ir 24,4 procentai. Moterims ir naujagimiams dažniausiai identifikuotas III ir Ia serotipo BGS. Bendras naujagimių įgimtos infekcijos dažnumas buvo 37,5 atvejai iš 1000 naujagimių, o BGS sukeltos infekcijos dažnumas - 3,6 atvejai iš 1000 naujagimių. Nebuvo nė vieno E.coli sukeltos ankstyvos naujagimių infekcijos atvejo. Klinikinis sepsis diagnozuotas 5 kartus dažniau nei mikrobiologiniais tyrimais... [toliau žr. visą tekstą] / Objective - to examine the prevalence of maternal and neonatal colonization of group B streptococcus (GBS) and Escherichia coli (E.coli) in our area, and to evaluate the colonization impact on early onset neonatal infections as a whole. Methods. A prospective cross-sectional study carried out at the Department of Obstetrics and Gynaecology and the Department of Neonatology of Kaunas University Hospital. Samples were collected from the lower vagina and the anorectum of pregnant women at 35-37 weeks of gestation or at delivery and the ear canal as well as throat of the neonates within 5 – 15 min of their lives. The distribution of serotypes of the GBS identified was determined using specific antisera and antimicrobial susceptibility was investigated by disc-diffusion method as described by National Committee for Clinical Laboratory Standards (NCCLS). Results. GBS carriage was detected in 148 (15.3%) of 970 women screened whereas E.coli colonisation was found in 193 (19.9%) of the women studied. The overall GBS and E.coli neonatal colonization rates were 6.4% and 14.4%; vertical transmission rates - 28.4% and 24.4%, respectively. The most common GBS serotypes were III and Ia. The overall incidence of early onset neonatal infection was estimated to be 37.5 per 1000 live birth and the incidence of early onset GBS disease in newborns – 3.6 per 1000 live birth. There was no case of early neonatal E. coli infection. The incidence of clinical sepsis in neonates was 5 fold higher that... [to full text]

Vaccination comes second in importance after introduction of clean water as a public health intervention which has largely contributed in the reduction of deaths from infectious diseases. Success in the development of a group A streptococcal (GAS) vaccine is expected to save 517 000 deaths per annum according to a recent independent review commissioned by the world health organization (WHO) and would offer an ideal means to prevent rheumatic heart disease (responsible for the greatest health burden) and other GAS-associated diseases which affect the health of 600 million. Traditional vaccine approaches (killed or live attenuated) have demonstrated great success against many bacterial and viral infectious diseases, crowned by the global eradication of smallpox announce by the WHO in 1980 and near-to-be announced eradication of polio viral disease. However, application of traditional techniques in many cases such as HIV/AIDS, malaria, GAS and Mycobacteria tuberculosis, has not shown the same success. Risk associated with the use of live–attenuated pathogens, such as recurrence of virulence (e.g. HIV), development of autoimmune diseases (e.g. GAS), and difficulties of manufacture hindered the use of such approaches. Other vaccine approaches such as subunit vaccines (recombinant proteins) and carrier conjugated vaccine are also hindered by the lack of suitable adjuvants, carriers and delivery systems. The current thesis focused on the design, synthesis and evaluation of novel adjuvants and vaccine delivery systems against GAS. The first chapter reviews recent approaches in the field of GAS vaccine design and new findings in immunology which represent the basis of our novel strategies. The second chapter describes the design, synthesis and evaluation of a novel library of lipopeptides as self-adjuvanting GAS vaccine candidates, composed of: (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2d) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment, or incorporation of P25 and J14 into a lipid-core peptide system (LCP) on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side-chain. Mucosal surface of the nasal-oral route is a primary site of GAS infections. An ideal GAS vaccine would have to elicit both mucosal as well as systemic immune responses and hence would not only prevent the development of GAS-associated diseases but also would prevent primary GAS infections. Therefore, the nasal route is considered a highly promising route of vaccine administration to provide local as well as systemic immune responses against pathogens that utilize mucosal surface as site of infection. The third chapter includes immunological assessment of the lipopeptide vaccine library described in the second chapter following intranasal immunization of B10BR (H-2k) mice. The whole library was first investigated in a small scale experiment (5 mice per group) to select promising candidates which demonstrate the best local and systemic J14-specific antibodies. Four selected lipopeptides were further investigated in a larger scale experiment (15 mice per group) followed by intranasal challenge of vaccinated mice with a virulent GAS M1 strain. The best local and systemic immune responses were demonstrated by a lipopeptide featuring a lipid moiety consisting of two 16 carbon chains incorporated at the C-terminus of the lipopeptide. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide candidate featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side-chain. A possible explanation for these results was investigated where antibodies elicited by the former candidate was found to better recognize the minimal B-cell epitope in the native p145 sequence of the M protein. Circular dichroism study of lipopeptides used in the previous experiment demonstrated that the former candidate features α-helical conformation which is required to produce protective J14-specific antibodies. Further studies are needed to explain structural features required to achieve both α-helicity and strong mucosal immune responses shown by the previously mentioned two lipopeptides. Signaling through toll-like receptors expressed by immune cells was recently shown to result in a robust immune response and was investigated as a possible mode of action for our novel lipopeptides. The fourth chapter introduces our lipopeptide vaccine approach as novel synthetic ligands targeting TLR2. A lipid moiety consisting of two alkyl chains of 16 carbons was found to achieve optimal TLR2 signaling regardless of the position of lipid attachment. Carbohydrates as polyhydroxy compounds provide an easily accessible class of compounds to design scaffolds (carriers) to attach lipids and peptide epitopes in different number and stereochemical positions which makes glycolipopeptides an attractive target for adjuvant research and structure-adjuvanticity relationships studies. The Fifth chapter reports immunological assessment of two series of glycolipopeptides as GAS vaccine candidates and novel vaccine delivery systems. The first series: lipid carbohydrate core peptide system (LCCP); represents a modification of the classical LCP system where polylysine dendrimer is replaced by different monosccharides as carriers for peptide antigens. LCCP analogues induced proper humoral immune responses against incorporated epitopes comparable to the LCP delivery system and as strong as the immune response elicited by CFA mixtures. Moreover, LCCP delivery system has been proved to be tolerant to the use of different epitopes as well as changing carbohydrate cores. Design of novel carbohydrate cores with different orthogonal protecting groups is needed to explore the potential advantage of various stereochemical arrangements provided by monosaccharides. The second series of glycolipopeptides incorporates various glycolipid moieties (self-adjuvanting activity) covalently coupled to the N-terminus of J8 (a model epitope). The new glycolipopeptide vaccine candidates (containing only one copy of J8) bear comparison with an LCP analogue (containing four copies of J8) which would improve the ease of synthesis, purification and cost of vaccine production. The slight difference in immunogenicity among these glycolipopeptides was difficult to be explained due to intervening effects of both the number and orientation of lipids on immunological activity. Further investigation is needed to determine the contribution of each factor.

Vaccination comes second in importance after introduction of clean water as a public health intervention which has largely contributed in the reduction of deaths from infectious diseases. Success in the development of a group A streptococcal (GAS) vaccine is expected to save 517 000 deaths per annum according to a recent independent review commissioned by the world health organization (WHO) and would offer an ideal means to prevent rheumatic heart disease (responsible for the greatest health burden) and other GAS-associated diseases which affect the health of 600 million. Traditional vaccine approaches (killed or live attenuated) have demonstrated great success against many bacterial and viral infectious diseases, crowned by the global eradication of smallpox announce by the WHO in 1980 and near-to-be announced eradication of polio viral disease. However, application of traditional techniques in many cases such as HIV/AIDS, malaria, GAS and Mycobacteria tuberculosis, has not shown the same success. Risk associated with the use of live–attenuated pathogens, such as recurrence of virulence (e.g. HIV), development of autoimmune diseases (e.g. GAS), and difficulties of manufacture hindered the use of such approaches. Other vaccine approaches such as subunit vaccines (recombinant proteins) and carrier conjugated vaccine are also hindered by the lack of suitable adjuvants, carriers and delivery systems. The current thesis focused on the design, synthesis and evaluation of novel adjuvants and vaccine delivery systems against GAS. The first chapter reviews recent approaches in the field of GAS vaccine design and new findings in immunology which represent the basis of our novel strategies. The second chapter describes the design, synthesis and evaluation of a novel library of lipopeptides as self-adjuvanting GAS vaccine candidates, composed of: (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2d) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment, or incorporation of P25 and J14 into a lipid-core peptide system (LCP) on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side-chain. Mucosal surface of the nasal-oral route is a primary site of GAS infections. An ideal GAS vaccine would have to elicit both mucosal as well as systemic immune responses and hence would not only prevent the development of GAS-associated diseases but also would prevent primary GAS infections. Therefore, the nasal route is considered a highly promising route of vaccine administration to provide local as well as systemic immune responses against pathogens that utilize mucosal surface as site of infection. The third chapter includes immunological assessment of the lipopeptide vaccine library described in the second chapter following intranasal immunization of B10BR (H-2k) mice. The whole library was first investigated in a small scale experiment (5 mice per group) to select promising candidates which demonstrate the best local and systemic J14-specific antibodies. Four selected lipopeptides were further investigated in a larger scale experiment (15 mice per group) followed by intranasal challenge of vaccinated mice with a virulent GAS M1 strain. The best local and systemic immune responses were demonstrated by a lipopeptide featuring a lipid moiety consisting of two 16 carbon chains incorporated at the C-terminus of the lipopeptide. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide candidate featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side-chain. A possible explanation for these results was investigated where antibodies elicited by the former candidate was found to better recognize the minimal B-cell epitope in the native p145 sequence of the M protein. Circular dichroism study of lipopeptides used in the previous experiment demonstrated that the former candidate features α-helical conformation which is required to produce protective J14-specific antibodies. Further studies are needed to explain structural features required to achieve both α-helicity and strong mucosal immune responses shown by the previously mentioned two lipopeptides. Signaling through toll-like receptors expressed by immune cells was recently shown to result in a robust immune response and was investigated as a possible mode of action for our novel lipopeptides. The fourth chapter introduces our lipopeptide vaccine approach as novel synthetic ligands targeting TLR2. A lipid moiety consisting of two alkyl chains of 16 carbons was found to achieve optimal TLR2 signaling regardless of the position of lipid attachment. Carbohydrates as polyhydroxy compounds provide an easily accessible class of compounds to design scaffolds (carriers) to attach lipids and peptide epitopes in different number and stereochemical positions which makes glycolipopeptides an attractive target for adjuvant research and structure-adjuvanticity relationships studies. The Fifth chapter reports immunological assessment of two series of glycolipopeptides as GAS vaccine candidates and novel vaccine delivery systems. The first series: lipid carbohydrate core peptide system (LCCP); represents a modification of the classical LCP system where polylysine dendrimer is replaced by different monosccharides as carriers for peptide antigens. LCCP analogues induced proper humoral immune responses against incorporated epitopes comparable to the LCP delivery system and as strong as the immune response elicited by CFA mixtures. Moreover, LCCP delivery system has been proved to be tolerant to the use of different epitopes as well as changing carbohydrate cores. Design of novel carbohydrate cores with different orthogonal protecting groups is needed to explore the potential advantage of various stereochemical arrangements provided by monosaccharides. The second series of glycolipopeptides incorporates various glycolipid moieties (self-adjuvanting activity) covalently coupled to the N-terminus of J8 (a model epitope). The new glycolipopeptide vaccine candidates (containing only one copy of J8) bear comparison with an LCP analogue (containing four copies of J8) which would improve the ease of synthesis, purification and cost of vaccine production. The slight difference in immunogenicity among these glycolipopeptides was difficult to be explained due to intervening effects of both the number and orientation of lipids on immunological activity. Further investigation is needed to determine the contribution of each factor.

Résumé : Le streptocoque de groupe B (SGB) est une bactérie commensale présente dans le tractus génito-urinaire de 10 à 30 % des femmes enceintes en santé. Ce pathogène est responsable de chorioamnionite, associée aux naissances prématurées et aux dommages cérébraux du nouveau-né. Les infections durant la grossesse, la chorioamnionite et la prématurité sont associées au développement de troubles du spectre de l’autisme. Notre hypothèse est qu’une exposition subclinique au SGB induit une réponse inflammatoire maternofoetale, menant à des troubles neurodéveloppementaux et comportementaux de type autistique dans la progéniture. L’objectif principal est d’étudier, à l’aide d’un nouveau modèle animal (rat) préclinique, les impacts d’une exposition au SGB en période prénatale sur le développement cérébral de la progéniture. Les rates Lewis gestantes sont injectées au jour de gestation 19 avec une dose de SGB de sérotype Ia (108 UFC/100µl) ou de saline. La réponse inflammatoire placentaire est caractérisée par immunohistochimie. Des tests comportementaux sont effectués entre les jours postnataux 7 et 40 afin d’évaluer la communication, le comportement exploratoire, l’intégration sensorielle et les interactions sociales. Une chorioamnionite dichotomique selon le genre est observée dans les placentas exposés au SGB, via une infiltration de cellules polymorphonucléaires. Cette infiltration est significativement plus proéminente dans les placentas associés aux fœtus mâles que ceux des fœtus femelles. Les mâles exposés au SGB ont un amincissement de la substance blanche cérébrale adjacente aux ventricules latéraux élargis. La progéniture mâle exposée au SGB présente des anomalies comportementales associées aux traits cardinaux des troubles du spectre de l’autisme, soit des déficits au niveau de la communication, des interactions sociales, du traitement de l’information sensorielle ainsi qu’au niveau d’autres comorbidités classiques de l’autisme, comme l’hyperactivité. Ces données démontrent pour la première fois que l’activation immune maternelle induite par l’infection au SGB joue un rôle dans l’induction d’anomalies neurodéveloppementales récapitulant celles observées chez les patients autistes, incluant la dichotomie de genre et le phénotype neurocomportemental. Ces résultats fournissent de nouvelles évidences en faveur du rôle dans la physiopathologie de l’autisme d’un facteur environnemental commun, et modifiable, d’inflammation gestationnelle. / Abstract : Group B Streptococcus (GBS) is a commensal bacterium present in the vagina of 10 to 30% of healthy pregnant women. GBS is responsible for chorioamnionitis, which can cause preterm birth and cerebral injuries in the newborn most often in the absence of maternofetal pathogen translocation. Maternal infection, chorioamnionitis and preterm birth are associated to autism spectrum disorders (ASD) in the progeny. Our hypothesis is that GBS-induced gestational infection induces a maternofetal inflammatory response leading to neurodevelopmental impairments and ASD-like behaviour in the offspring. Our goal was to study, with a new preclinical animal model, the impacts of GBS-induced gestational inflammation on the neurodevelopmental features in the offspring. We characterized GBS-induced placental and neurobehavioural outcomes. Dams were exposed at gestational day 19 to live GBS or saline. The placental inflammatory response was studied by immunohistochemistry. Behavioural tests were performed between postnatal days 7 and 40 to assess communication, exploratory abilities, sensory integration and social interactions. GBS-exposed placentas displayed chorioamnionitis featured by infiltration of polymorphonuclear cells, which was significantly more prominent in males than in females. GBS-exposed males showed a reduced thickness of periventricular white matter. Male offspring exposed to GBS had early onset of cardinal ASD-like traits affecting social interaction, communication (ultrasonic vocalizations), treatment of sensory information (prepulse inhibition), preference toward mother cue (nest-seeking), and some other classic ASD comorbidities such as hyperactivity (open field). Overall, these data show for the first time that maternal immune activation due to live GBS plays a key role in the induction of neurodevelopmental abnormalities recapitulating those of human ASD, including gender dichotomy and neurobehavioural phenotype. These results provide new evidence in favor of the role of a common and modifiable infectious/inflammatory environmental factor in human ASD pathophysiology.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Streptococcus agalactiae, ou Streptococcus do grupo B (GBS), é um importante patógeno oportunista que causa pneumonia, sepse e meningite em recém-nascidos e infecções em adultos imunocomprometidos. O pulmão aparentemente é o portal de entrada para o EGB na corrente sanguínea o que pode evoluir para uma septicemia. Os mecanismos de virulência relevantes envolve a habilidade do EGB em penetrar e sobreviver intracelularmente em células hospedeiras. Neste trabalho, foram analisados os mecanismos moleculares da apoptose epitelial induzida pelo EGB, e a produção de óxido nítrico (NO) e espécies reativas de oxigênio (ROS) em células epiteliais respiratórias A549 durante a infecção por EGB. Todas as amostras de EGB exibiram a capacidade de aderir e invadir células A549. A sobrevivência intracelular do EGB em células A549 ocorreu durante 24 h de incubação sem replicação do patógeno. No entanto, a amsotra 88641-V isolada de vagina não sobreviveu após 0,5 h de interação. O EGB promoveu a perda de viabilidade do epitélio durante a infecção. As alterações morfológicas em células A549 infectadas com o EGB incluem arredondamento celular, condensação nuclear, encolhimento celular e perda de contato célula-célula e célula-substrato. A dupla marcação AV/IP revelou que amostras de EGB sorotipo III induziram apoptose enquanto amostras do sorotipo V induziram morte celular semelhante a necrose em células A549. Caspase-3 foi ativada durante a apoptose induzida por EGB em células epiteliais. No entanto, a ativação de caspases-8 e -9 foi detectada apenas para a amostra 88641-V e as amostras EGB do sorotipo III, respectivamente. Experimentos comparativos de Immunoblotting revelaram que o EGB induziu um aumento da expressão Bim, uma proteína pró-apoptótica e diminuiu a expressão de Bcl-2 e Bcl-xL, proteínas anti-apoptóticas. As células A549 apresentaram perda de potencial de membrana mitocondrial &#916;&#968;m e co-localização com o Bax. Ensaio de espectrometria de massa identificou a proteína PI-2a, uma proteína estrutural de pili, que exibe atividade carboxipepdidase. Descobrimos que os dois sorotipos (III e V) induziram a produção ROS e NO em células A549. Em conclusão, a apoptose induzida pelo EGB em células A549 é um mecanismo importante de virulência, resultando na destruição de tecidos, escape do sistema imune do hospedeiro com espalhamento bacteriano e, em consequência, a doença invasiva ou uma infecção sistémica. / Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. The lung is the apparent portal of entry for GBS into the bloodstream, after which septicemia may ensue. A relevant virulence mechanism involves the ability of GBS to penetrate and to survive intracellularly within these host cells. In this work, we analyzed the molecular mechanisms of GBS-induced epithelial apoptosis, and nitric oxide (NO) and reactive oxygen species (ROS) production by lung epithelial cell line A549 cells during infection with GBS. All GBS exhibited the ability to adhere and to invade A549 cells. The survival of GBS within A549 cells without replication was shown during 24 h incubation. However, the 88641-V strain isolated from vagina did not survive after 0.5 h of interaction. GBS promoted the loss of viability of the epithelium during infection. The morphological changes in A549 cells infected with GBS included cell rounding, nuclear condensation, cellular shrinkage and loss of cell-cell contact and cell-substrate. The double staining AV / IP revealed that GBS serotype III induced apoptosis while GBS serotype V induced like necrosis cell death in A549 cells. Caspase-3 was activated during GBS-induced endothelial apoptosis. However, activation of caspases-8 and -9 was detected only by GBS 88641-V and GBS-III, respectively. Comparative immunoblotting experiments revealed that GBS induced an increasing pro-apoptotic Bim expression and decreasing anti-apoptotic Bcl-2 and Bcl-XL expression. A549 cells exhibited loss of mitochondrial membrane potential &#916;&#968;m with Bax colocalization. Mass spectrometry assay identified protein PI-2a, a structural protein pili, which exhibit carboxipepdidase activity. We found that both serotypes (III and V) induced ROS and NO production in A549 cells. In conclusion, apoptosis of A549 cell induced by GBS is an important virulence mechanism resulting in tissue destruction, escape from the host immune system with bacterial spreading and, in consequence, invasive disease or systemic infection.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Streptococcus agalactiae, ou Streptococcus do grupo B (GBS), é um importante patógeno oportunista que causa pneumonia, sepse e meningite em recém-nascidos e infecções em adultos imunocomprometidos. O pulmão aparentemente é o portal de entrada para o EGB na corrente sanguínea o que pode evoluir para uma septicemia. Os mecanismos de virulência relevantes envolve a habilidade do EGB em penetrar e sobreviver intracelularmente em células hospedeiras. Neste trabalho, foram analisados os mecanismos moleculares da apoptose epitelial induzida pelo EGB, e a produção de óxido nítrico (NO) e espécies reativas de oxigênio (ROS) em células epiteliais respiratórias A549 durante a infecção por EGB. Todas as amostras de EGB exibiram a capacidade de aderir e invadir células A549. A sobrevivência intracelular do EGB em células A549 ocorreu durante 24 h de incubação sem replicação do patógeno. No entanto, a amsotra 88641-V isolada de vagina não sobreviveu após 0,5 h de interação. O EGB promoveu a perda de viabilidade do epitélio durante a infecção. As alterações morfológicas em células A549 infectadas com o EGB incluem arredondamento celular, condensação nuclear, encolhimento celular e perda de contato célula-célula e célula-substrato. A dupla marcação AV/IP revelou que amostras de EGB sorotipo III induziram apoptose enquanto amostras do sorotipo V induziram morte celular semelhante a necrose em células A549. Caspase-3 foi ativada durante a apoptose induzida por EGB em células epiteliais. No entanto, a ativação de caspases-8 e -9 foi detectada apenas para a amostra 88641-V e as amostras EGB do sorotipo III, respectivamente. Experimentos comparativos de Immunoblotting revelaram que o EGB induziu um aumento da expressão Bim, uma proteína pró-apoptótica e diminuiu a expressão de Bcl-2 e Bcl-xL, proteínas anti-apoptóticas. As células A549 apresentaram perda de potencial de membrana mitocondrial &#916;&#968;m e co-localização com o Bax. Ensaio de espectrometria de massa identificou a proteína PI-2a, uma proteína estrutural de pili, que exibe atividade carboxipepdidase. Descobrimos que os dois sorotipos (III e V) induziram a produção ROS e NO em células A549. Em conclusão, a apoptose induzida pelo EGB em células A549 é um mecanismo importante de virulência, resultando na destruição de tecidos, escape do sistema imune do hospedeiro com espalhamento bacteriano e, em consequência, a doença invasiva ou uma infecção sistémica. / Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. The lung is the apparent portal of entry for GBS into the bloodstream, after which septicemia may ensue. A relevant virulence mechanism involves the ability of GBS to penetrate and to survive intracellularly within these host cells. In this work, we analyzed the molecular mechanisms of GBS-induced epithelial apoptosis, and nitric oxide (NO) and reactive oxygen species (ROS) production by lung epithelial cell line A549 cells during infection with GBS. All GBS exhibited the ability to adhere and to invade A549 cells. The survival of GBS within A549 cells without replication was shown during 24 h incubation. However, the 88641-V strain isolated from vagina did not survive after 0.5 h of interaction. GBS promoted the loss of viability of the epithelium during infection. The morphological changes in A549 cells infected with GBS included cell rounding, nuclear condensation, cellular shrinkage and loss of cell-cell contact and cell-substrate. The double staining AV / IP revealed that GBS serotype III induced apoptosis while GBS serotype V induced like necrosis cell death in A549 cells. Caspase-3 was activated during GBS-induced endothelial apoptosis. However, activation of caspases-8 and -9 was detected only by GBS 88641-V and GBS-III, respectively. Comparative immunoblotting experiments revealed that GBS induced an increasing pro-apoptotic Bim expression and decreasing anti-apoptotic Bcl-2 and Bcl-XL expression. A549 cells exhibited loss of mitochondrial membrane potential &#916;&#968;m with Bax colocalization. Mass spectrometry assay identified protein PI-2a, a structural protein pili, which exhibit carboxipepdidase activity. We found that both serotypes (III and V) induced ROS and NO production in A549 cells. In conclusion, apoptosis of A549 cell induced by GBS is an important virulence mechanism resulting in tissue destruction, escape from the host immune system with bacterial spreading and, in consequence, invasive disease or systemic infection.

Orientador: Renato Passini Junior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T01:25:35Z (GMT). No. of bitstreams: 1 Nomura_MarceloLuis_D.pdf: 838174 bytes, checksum: a795b39457b62a18ecb17c4609ece12d (MD5) Previous issue date: 2004 / Resumo: Objetivos: Identificar a taxa de prevalência e fatores de risco de colonização materna por estreptococo do grupo B (EGB) em gestantes com trabalho de parto prematuro (TPP) e/ou ruptura prematura pré-termo de membranas (RPM). Métodos: Foram colhidos dois swabs anais e vaginais de 203 gestantes atendidas no CAISM-UNICAMP. Um swab de cada local foi colocado em meio de transporte e enviados para cultura em placas de ágar-sangue, os outros dois foram incubados por 24 horas em meio de Todd-Hewitt para posterior semeadura em placas de ágar-sangue. Resultados: A prevalência de colonização materna por EGB foi de 27,6% (56 gestantes). As taxas de colonização por diagnóstico foram 34,7% para RPM, 25,2% para TPP e 17,8% para TPP + RPM. As variáveis raça branca, baixo nível de escolaridade e infecção urinária foram associadas a maiores taxas de colonização na análise multivariada. A presença de infecção urinária foi a única variável significativamente associada à colonização materna na análise multivariada. A taxa de detecção do estreptococo do grupo B foi significativamente maior com o uso do meio seletivo e com a associação de coleta de culturas anais e vaginais. A taxa de colonização neonatal foi de 3,1%. Ocorreram dois casos de sepse precoce por EGB nesta amostra, com prevalência estimada de 10,8 casos por mil nascidos vivos e mortalidade de 50%. Conclusão: A amostra avaliada apresenta altas taxas de colonização materna por Streptococcus agalactiae. É necessário o uso de meio de cultura seletivo e a associação de culturas anorretais e vaginais para aumentar a taxa de detecção do EGB. A incidência de sepse neonatal precoce foi elevada nesta população / Abstract: Objective: to study group B streptococcus maternal colonization rates and risk factors in women with preterm labor (PTL) and preterm premature rupture of membranes (PROM). Methods: Vaginal and anal swabs (two of each) were colected from 203 women followed at CAISM-UNICAMP. One of each swab was placed in transport media and then cultured in blood-agar plates, the other swabs were incubated in Todd-Hewitt selective media for 24 hours and then subcultured in blood-agar plates. Results: Maternal colonization rate was 27.6% (56 women). Colonization rates by admission diagnosis were 34.7% in PROM, 25.2% in PTL and 17.8% in PTL and PROM. White race, less than elementary education level and urinary tract infection were associated with maternal colonization in the univariate analysis. Urinary tract infection was the only variable associated with maternal colonization in a multivariate analysis. GBS detection rates were significantly higher with the use of selective culture media and with sampling of both vaginal and anorectal sites. Neonatal colonization rate was 3.1%. There were two cases of early-onset neoanatal sepsis caused by GBS, with an estimated prevalence of 10.8 cases per thousand live borns and a mortality rate of 50%. Conclusions: This sample of women had high GBS colonization rates. The use of selective culture media and collection of both anal and vaginal samples is necessary in order to maximize GBS detection rates. Early-onset neonatal sepsis incidence was high in this population / Doutorado / Tocoginecologia / Doutor em Tocoginecologia