Thesis (Ph. D.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains x, 185 p. : ill. Includes abstract. Includes bibliographical references.

Autoimmune encephalitic (AE) syndromes represent a unique manifestation of autoimmunity: the immune system recognizes the brain as foreign, and interferes with neuronal function. AE syndromes are characterized by hallucinations, paranoia, anxiety, seizures, and autonomic dysregulation, and progress over a matter of weeks as autoantibodies targeting the brain bind more densely to their CNS targets. Development of AE has been linked to peripheral tumors and infection, both of which provide structural mimetics to CNS antigens to incite an immune response in the periphery. How these brain-specific antibodies reach the CNS remains unclear. In rare cases, Group A Streptococcus (GAS, S. pyogenes) infections can cause CNS autoimmunity targeting the basal ganglia, termed post-infectious basal ganglia encephalitis (BGE), manifesting as motor (Sydenham’s chorea) and psychiatric (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus, PANDAS) abnormalities. In a mouse model of post-infectious BGE, we have shown that repeated intranasal GAS infections generate robust T cell infiltration into the CNS, concomitant with blood-brain barrier breakdown, microglial activation, and olfactory synapse degradation. However, the different T cell subtypes that enter the brain, how they enter the CNS, and their relative contributions to neural dysfunction and neuroinflammation remain unclear. Th17 lymphocytes are heavily implicated in many autoimmune diseases, but whether the inflammatory post-infectious BGE reaction induces functional deficits in odor processing and requires Th17 lymphocytes was unknown. Here we demonstrate that mice lacking Th17 lymphocytes display both reduced BBB impairment and antibody infiltration. Furthermore, multiple GAS infections induce deficits in odor processing, which are partially ameliorated in Th17 cell-deficient mice. Notably, neuroinflammation and some excitatory synaptic loss persist, due to the presence of Th1 lymphocytes. Th17 lymphocytes are therefore critical for both selective CNS entry of autoantibodies and neural circuit impairment during post-infectious BGE. By examining the regulation of chemokine ligand expression in GAS-inoculated mice, we determined that CCL20/CCR6 and CCL2/CCR2 chemokine axes are implicated in T cell homing to the brain. In chemokine receptor mutant CCR6+/- CCR2-/- mice, T cell infiltration is reduced by 86.2%, and microglial activation is blunted. These findings suggest that CCL2/CCR2 and CCL20/CCR6 signaling may play a role in T cell homing to the brain and neuroinflammation. Finally, we first assessed behavioral and immune responses in two different GAS exposure models. It was clear that while behavioral abnormalities can be recapitulated in mice given subcutaneous GAS immunizations, this elicited relatively weak cellular and humoral immune responses. By contrast, mice given intranasal GAS inoculations showed minimal behavioral abnormalities, but elicited robust humoral and cellular immune responses. Taken together, these data demonstrate the pivotal role of Th17 lymphocytes in brain pathology and olfactory processing deficits after recurrent GAS infections in our mouse model. Our intranasal inoculation model supports the conclusion that post-infectious BGE is autoimmune in nature, despite the absence of behavioral symptoms in this model. Using multiple mouse models of post-infectious BGE may allow us to study distinct facets of disease pathogenesis. Finally, this work underscores the ability of T cells to incite neuroinflammation, provides a useful clinical diagnostic test in olfactory functional assessments, and lends support to T cell immunotherapy strategies in patients with post-infectious BGEs.

Streptomycin resistance in both gram-positive and gram-negative bacteria is usually caused by a single mutation in the rpsL gene. The rpsL gene encodes the S12 protein of the ribosomal complex. The rpsL genes of various bacteria have consensus regions in their sequences. Primers were designed from these consensus pockets and a fragment of the rpsL gene was sequenced from S. gordonii using PCR based methodologies. Using the Multiplex Restriction Sequence PCR(mRS PCR), which used the known primer at one end and a restriction site primer on the other, a gene walk was conducted. In streptomycin resistant strains of S. gordonii, namely GP204, SP204 and SP635, the AAA coding for Lys56 was mutated to ACA, coding for Thr56. The lysine to threonine transition, causing resistance to streptomycin was identical to that expected from the literature. The streptomycin resistance gene of S. pyogenes was mapped using similar techniques. Streptomycin resistant strains S43 ATCC, 543/192/4 and S43/192/30R were studied. In streptomycin resistant S43 ATCC and S43/192/30R strains, the lysine 56 changed to isoleucine and threonine respectively. Surprisingly, the 192/4 had two mutations, in each of the two hotspots in the rpsL gene where mutations due to streptomycin resistance occur. It had the amino acid 56, lysine, mutated to arginine and lysine 101 changed to asparagine. To check if this mutation was stable in the host animal, S43/192/4 P8 (S43/192/4 passaged eight times in mice) was sequenced and the sequence was identical to the streptomycin resistant 192/4. Hence, the lys101 mutation was stable and unlike the ancillary mutations in E.coli and S. typhimurium, which are compensated by new mutations. The pathogenesis of S. pyogenes depends in part on the ability of the pathogen to adhere to the epithelial cells of the throat and the quantity of M protein. Pathogenesis studies done on mice revealed the avirulence of S43/192/4smR strain. To elucidate the reason for this avirulence, the adherence properties and the production of M protein of the two strains S43/192/4smR and S43/192/30R were tested. Qualitative immunoblot analysis of the M protein of 192/4 and 30R revealed no significant difference. Competition ELISA was conducted to quantitate the M protein, and this also did not show any significant difference in the M protein levels. The adherence of 30R and 192/4 was measured on human pharyngeal epithelial cell line. The adherence properties of S43/192/4 SmR, was no different from other strains in this experiment. Electron microscopy, using immunogold to highlight the M protein on the cell surface showed no differences. / Graduation date: 2000

No

Streptococcus pyogenes és un patogen humà responsable d'un ampli ventall d'infeccions que varien des d'infeccions superficials com faringitis i impetigen a formes sistèmiques greus com fascitis necrosant (FN) i síndrome del xoc tòxic estreptocòccic (SSTS). El ressorgiment i persistència de formes invasives greus descrit des de mitjans dels anys 1980 ha motivat una intensa recerca sobre els aspectes epidemiològics, microbiològics i clínics d'aquestes infeccions. S'ha realitzat un estudi retrospectiu de base hospitalària que inclou 126 soques de S. pyogenes (27 procedents d'infeccions invasives i 99 d'infeccions no invasives) aïllades entre gener de 1999 i juny de 2003. Les soques de S. pyogenes es van caracteritzar en base a la distribució de tipus i subtipus emm i els perfils genètics de superantígens (SAgs) (speA-C, speF-J, speL, speM, ssa i smeZ). Tanmateix, es va determinar la prevalença i els mecanismes de resistència a macròlids, tetraciclina i levofloxacino. Les formes clíniques més freqüents d'infecció invasiva van ser les infeccions de la pell i teixits tous (40,7%). La SSTS es va registrar en quatre (14,8%) dels casos invasius i es va associar a FN en la meitat dels casos. La majoria dels pacients afectats de quadres invasius eren adults, en particular d'edat avançada i de mitjana edat, i una elevada proporció presentaven factors predisposants, destacant l'alteració de la barrera cutània, la infecció per HIV, l'ús de drogues per via parenteral, i les neoplàsies. En la col·lecció de 126 soques analitzada es van identificar un total de 29 tipus emm amb una distribució encapçalada pel tipus emm1 (17,5%), seguit d'emm3 (8,7%), emm4 (8,7%), emm12 (7,1%), emm28 (7,1%), emm11 (6,3%) i emm77 (6,3%). Aquests set tipus van constituir el 61,9% del total de soques. No es van observar diferències significatives en la distribució de tipus emm entre soques aïllades d'infeccions invasives i no invasives amb l'única excepció del tipus poc freqüent emm25 que es va trobar associat a infeccions invasives en addictes a drogues per via parenteral. Es va trobar una forta correlació entre el patró de SAgs i el tipus emm independentment del tipus d'infecció. La resistència a eritromicina va mostrar un increment anual progressiu del 16,6% (1999) al 38,8% (2003) i va estar causada per soques pertanyents a 11 tipus emm. Les soques mef(A) positives dels tipus emm4, emm12 i emm75 i erm(B) positives dels tipus emm11 i emm25 constituïren el 80% de les soques resistents. La freqüència de resistència a tetraciclina va fluctuar durant el període estudiat (màxim 34,6% el 2002 i mínim 15,8% el 2001) i va ser superior en les soques resistents a eritromicina que en les soques sensibles (42,8% vs 18,7%). En les soques resistents a tetraciclina el gen tet(M) va ser el predominant i es va trobar en soques pertanyents a 14 tipus emm, mentre que el gen tet(O) només es va trobar en soques emm77. No es van observar diferències significatives en la prevalença de resistència a eritromicina ni a tetraciclina en el grup invasiu respecte del no invasiu. La prevalença de resistència a levofloxacino fou del 3,2%, incloent quatre soques amb sensibilitat reduïda o resistència intermèdia (CIM 2-4 µg/ml) i dues soques amb resistència d'alt nivell (CIM >32 µg/ml). La resistència de baix nivell es va associar a substitucions únicament en ParC (Ser80Pro, Ser79Ala, Ser79Phe i Ala121Val), mentre que la resistència d'alt nivell es va relacionar amb mutacions en ParC (Ser79Phe i Ala121Val) i GyrA (Ser81Tyr). / Streptococcus pyogenes (GAS) is a human pathogen responsible for a wide array of infections, ranging from pharyngitis and impetigo to severe invasive infections such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The resurgence and persistence of severe forms of GAS diseases reported since the mid 1980s have motivated intensive research on epidemiological, microbiological and clinical aspects of these diseases. A retrospective hospital-based study was conducted including 126 GAS isolates (27 from invasive infections and 99 from non-invasive infections) collected from January 1999 to June 2003. GAS isolates were characterized by emm type and subtype and superantigen (SAg) gene profile (speA-C, speF-J, speL, speM, ssa and smeZ). The prevalence and mechanisms of macrolide, tetracycline and levofloxacin resistance were also determined. The most common clinical presentations of invasive cases were skin and soft-tissue infections (40.7%). SSTS occurred in four cases (14.8%) and was associated to NF in half of the cases. Most invasive cases were found in adults, in particular among the elderly and the middle-aged, and a large proportion had underlying conditions, the most frequent being skin lesions, HIV infection, injection drug use, and malignancy. A total of 29 emm types were identified among the 126 isolates; the most prevalent were emm1 (17.5 %), followed by emm3 (8.7 %), emm4 (8.7 %), emm12 (7.1 %), emm28 (7.1 %), emm11 (6.3 %) and emm77 (6.3 %). These seven emm types accounted for 61.9 % of isolates. There were no differences in the emm type distribution between invasive and non-invasive infections, except for emm25 isolates, which were associated with invasive infections in injecting drug users. The SAg gene profiles were closely associated with the emm type and were independent of the disease type. The prevalence of erythromycin resistance showed an annual progressive increase from 16.6% (1999) to 38.8% (2003) and was caused by isolates belonging to 11 emm types. mef(A)-positive emm types 4, 12 and 75, and erm(B)-positive emm types 11 and 25 were responsible for up to 80% of the erythromycin-resistant isolates. The prevalence of tetracycline resistance fluctuated over the period studied (maximum 34.6% in 2002 and minimum 15.8% in 2001) and was higher in erythromycin-resistant isolates than in susceptible isolates (42.8% vs 18.7%). Among the tetracycline-resistant isolates, the tet(M) determinant was the most prevalent and was distributed in isolates belonging to 14 emm types, whereas tet(O) was only found in emm77 isolates. No significant differences in resistance rates to erythromycin or tetracycline were found between invasive and non-invasive isolates. The rate of resistance to levofloxacin was 3.2%, encompassing four isolates with reduced susceptibility or intermediate resistance (MIC 2-4 µg/ml) and two isolates with a high level of resistance (MIC >32 µg/ml). Low-level resistance was associated with alterations in ParC (Ser80Pro, Ser79Ala, Ser79Phe and Ala121Val), while high-level resistance was associated with alterations involving both ParC (Ser79Phe and Ala121Val) and GyrA (Ser81Tyr).

Zugl.: Aachen, Techn. Hochsch., Diss., 2008

Group A Streptococcus (GAS) is responsible for mild and common infections like tonsillitis and pharyngitis, and more serious invasive disorders like necrotizing fasciitis and glomerulonephritis. The ability to invade tissues is closely linked to the virulence factors expressed by the bacterium. Hyaluronic acid capsule expression is variable among all the strains in S. pyogenes and confers the capacity to evade the immune response. In a previous study, it was found that capsule production in CovR mutants was temperature-regulated, showing a basal level of capsule production at 37℃ but increased production was observed at 25℃. Moreover, it was found that when CvfA, an endoribonuclease, is mutated, this thermoregulation is abolished. Since an antisense RNA was found spanning the entire capsule operon, another ribonuclease, RNaseIII which is involved in dsRNA digestion, was considered as a possible candidate to regulate the antisense and capsule transcripts. In this study, the objective is to find if and how the ribonucleases regulate the capsule transcript and antisense RNA. Data suggests that RNaseIII does not have a role in the regulation. On the other hand, CvfA showed a key role in regulating transcript levels. Furthermore, temperature appears to have some effect on its activity.

Streptococcus pyogenes is a human-specific pathogen that can cause a wide variety of diseases. These diseases range from the relatively mild pharyngitis and impetigo to invasive diseases such as necrotizing fasciitis to post-streptococcal sequelae such as rheumatic heart disease. The bacteria are frequently carried asymptomatically and may cause recurrent disease. Corresponding with their etiologic variation amongst diseases, clinical isolates demonstrate diverse virulence factor expression and random genetic mutations. In these studies, we examine the role of intracellular residence during survival as a niche for the diversification of S. pyogenes. Survival was previously studied using two in vitro systems: long-term stationary phase survival in culture and survival within epithelial cells in the presence of extracellular antibiotics. The surviving populations diversified, giving rise to stable strains with alternate colony morphologies, distinct proteomes, and altered metabolic properties. Further analysis in these studies showed that alterations in colony morphology were not solely observed during survival, but could also be induced in models mimicking acute infection. However, diversification in certain metabolic pathways occurred only during survival, and this metabolic diversification was observed at the transcriptional level. Further, one of three clinical isolates from patients with recurrent pharyngitis was altered in its metabolic profile, suggesting metabolic diversification may be occurring in vivo. The survivor strains had varied transcriptional changes in the genes encoding the virulence factors emm, slo, and speB. All of the stationary phase-derived survivor strains and two intracellular survival-derived strains had attenuated virulence in zebrafish. Most of the attenuated strains disseminated to the spleen and were cleared within three days. A whole blood killing assay showed a strong correlation between bacterial killing and emm expression. While the diversification appeared random, these strains retained their multilocus sequence type (MLST). These results suggest S. pyogenes strains with the same MLST, but diverse virulence properties, may arise during survival in the host. / Microbiology and Immunology

Thèse d'exercice : Médecine. Médecine générale : Paris 12 : 2006. / Titre provenant de l'écran-titre. Bibliogr. f. 66-69.

Les Streptocoques Béta-hémolytiques du groupe A (GAS) sont responsables de manifestations cliniques variées et de séquelles non suppuratives comme notamment le rhumatisme articulaire aigu (RAA). Les affections sévères à GAS tuent plus de 500.000 personnes chaque année. Le pouvoir pathogène du GAS est encore mal compris. Il semble être notamment lié à la présence de nombreux gènes codant pour des facteurs de virulence dans le génome du GAS, dont celui codant la protéine emm. La protéine transmembranaire M joue un rôle essentiel dans la virulence du GAS. Le typage moléculaire des GAS se base sur la séquence de la partie hypervariable de ce gène (emm-typing). L’épidémiologie du GAS semble varier au cours du temps et en fonction de la localisation géographique et/ou du contexte socio-économique. Cependant, les différences dans les critères d’inclusion des différentes études épidémiologiques disponibles dans la littérature rendent les comparaisons difficiles. <p>Pour mieux évaluer ces variations, nous avons mené une analyse prospective de l’épidémiologie clinique et moléculaire d’isolats de GAS provenant d’enfants présentant une infection à GAS, simultanément en deux localisations géographiques différentes (Bruxelles et Brasília, Brésil).<p>Un des points importants de notre étude a été la mise en évidence de la diversité génétique de la protéine M des isolats belges et brésiliens. Alors que de nombreux emm-types différents sont retrouvés à Brasília (48 emm-types sur 128 isolats), ceux retrouvés à Bruxelles sont relativement peu nombreux (20 emm-types sur 200 isolats) et sont ceux communément retrouvés dans les pays industrialisés. Afin de mieux comprendre les bases moléculaires de cette différence, une analyse phylogénétique basée sur la quasi-totalité de la séquence de la protéine M exposée à la surface de la bactérie a été réalisée. Cette analyse a permis de montrer que les emm-types belges sont génétiquement éloignés les uns des autres alors que les emm-types brésiliens sont génétiquement plus proches. De manière intéressante, cette analyse a montré que les souches belges présentent une grande diversité au niveau de la région de la protéine M dite ‘constante’. En conséquence, la diversité génétique globale des protéines M belges et brésiliennes est similaire, mais elle se situe dans des régions différentes de la protéine M, ce qui pourrait indiquer l’existence de pressions de sélection différentes entre les deux pays. D’un point de vue vaccinal, ces résultats indiquent qu’un vaccin dirigé contre certaines des parties constantes de M présenterait une bonne couverture théorique dans les deux pays. Par contre, le vaccin 26-valent, en cours d’évaluation clinique, aurait une couverture théorique de 76% à Bruxelles et de 32% à Brasília. <p>Notre analyse phylogénétique a également permis de montrer que la non-sensibilité à la ciprofloxacine (observée dans 22,5 % et 9% des souches belges et brésiliennes respectivement) survient dans des souches génétiquement éloignées, contrairement à ce qui est proposé actuellement dans la littérature. De plus, nous avons mis en évidence un polymorphisme au sein des gènes codant les topoisomérases cibles de la ciprofloxacine. L’identification de mutations responsables du phénotype de non-sensibilité nécessite par conséquent une confirmation expérimentale.<p>Les manifestations cliniques sont assez différentes entre Bruxelles et Brasília. Les infections cutanées sont beaucoup plus fréquentes à Brasília. De manière intéressante au Brésil, des souches de GAS présentant un tropisme cutané sont isolées du pharynx. Ces souches ‘cutanées’ pourraient avoir acquis des déterminants génétiques leur permettant de se développer dans des tissus pharyngés. De plus, ces résultats pourraient remettre en question le postulat que seules les souches de tropisme pharyngé sont impliquées dans le développement du RAA. D’autres études épidémiologiques dans des pays où le RAA est endémique devront être réalisées afin de préciser nos résultats et de mieux comprendre les mécanismes moléculaires menant au développement du RAA.<p>Cependant, étant donné la prévalence du RAA et l’accès limité au diagnostic microbiologique des pharyngites dans le réseau public de soins au Brésil, nous avons développé un score clinique permettant de limiter les traitements antibiotiques chez les enfants probablement atteints de pharyngites virales. L’utilisation de ce score permettrait de réduire le nombre de prescriptions antibiotiques dans les pharyngites de l’enfant de 41 à 55% à Brasília.<p>Le choc toxi-infectieux est une pathologie relativement rare et le RAA n’est quasi plus décrit dans les pays développés. Cependant, deux nourrissons ont présenté un choc toxi-infectieux suivi d’un RAA (HUDERF, Bruxelles). A notre connaissance, cette association clinique n’a jamais été décrite. L’analyse de ces deux cas du point de vue de la virulence bactérienne a révélé la présence de nombreux gènes de facteurs de virulence, portés par des phages et différents dans les deux souches. Nos résultats illustrent la complexité de la relation hôte-pathogène. <p>La capacité des bactéries à s’adapter à leurs hôtes et à causer des pathologies dépend de nombreux facteurs, qui varient d’un isolat à l’autre, et dont l’importance varie d’un hôte à l’autre. Notre travail a permis d’exemplifier la diversité génétique des GAS, aussi bien au niveau du gène emm qu’au niveau des facteurs de virulence, et de l’implication de ceux-ci dans le développement de pathologies streptococciques rares. <p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished