Helicobacter pylori is one of the most genetically diverse bacterial species that successfully colonizes at least 50% of the world population. It has been associated with humans for thousands of years and most probably evolved from ancestral gastric Helicobacter species in early mammals. One of the important characteristics of this pathogen is the degree of allelic diversity and genetic variability which helps it to adapt and colonize. Phase variation is one of the mechanisms used by H. pylori to generate variation. The presence of homopolymeric nucleotide or dinucleotide repeats in an ORF make it prone to frequent length changes as a consequence of slipped strand mispairing mediated mutagenesis. Interestingly, R-M genes comprise a significant percentage of H. pylori strain-specific genes and are more prevalent in H. pylori than in other bacterial species whose genomes have been fully sequenced. R-M systems in H. pylori have been identified on the basis of sequence similarity to known restriction endonucleases and methyltransferases, genetic organization, and specific enzyme isolation and characterization. Analysis of genome sequences of H. pylori strains 26695, J99, HPAGI and 26 others has revealed the presence of more than 20 R-M systems in each stain, which are far more than detected in any other bacterial genome sequence till date. hp1369 and hp1370 are two ORFs in stain 26695 coding for hypothetical proteins. hp 1369 has a stretch of poly-G repeats, thus making hp1369-hp1370, a candidate of phase variation. hpag1_1313 is homolog of hp1369-hp1370 which got up-regulated, in a person suffering from acute gastritis, thus making these genes an interesting subject of investigation. This study was therefore initiated with the following objectives: 1. Cloning, over-expression and purification of Type III MTase (ORF- hp1369- hp1370) and its cognate restriction enzyme (hp1371). 2. Biochemical characterization of MTase (HP1369-HP1370): Determination of oligomeric status, kinetic properties, binding affinities for AdoMet and DNA. Sequence analysis shows the presence of a poly-G track (10 Gs) at 3’-end of hp1369 which is a signature sequence for phase variation. Addition of a single nucleotide can place both hp1369 and hp1370 in-frame, which could code for a single polypeptide. hp1369 and hp1370 in H. pylori strain 26695 alone do not code for any functional protein but with the fusion of hp1369 and hp1370 can code for a protein with all the nine motifs of a DNA MTase. Interestingly, on the basis of arrangement of Motifs, it is probably the first example of ε type of methyltransferase. By site-directed mutagenesis a single G nucleotide was inserted in the poly-G track and both the ORFs (hp1369 and hp1370 ) became in-frame, coding for fully functional HP1369-HP1370 MTase. Kinetic parameters for functional HP1369-HP1370 MTase were determined, and has shown that there was substrate inhibition in methylation reaction at higher concentrations of AdoMets. When preincubation studies were done, enzyme-DNA complex was found to be more competent than enzyme-AdoMet complex. HP1369-HP1370 MTase exists as dimer in solution, having affinity for duplex DNA and does not bind to single-stranded DNA. Binding affinity for ligand (AdoMet) was determined by Isothermal Titration Calorimetry method. H. pylori has evolving restriction-modification systems. It is capable of taking new R-M systems from the environment in the form of DNA released from other bacteria or other Helicobacter strains. H. pylori genome is dynamic with high mutation rates. Random mutations in R-M genes can result in a non-functional R-M systems or R-M systems with new properties. The dynamics of R-M system plays a vital role in shaping up the genome.

More than half of all people worldwide are infected with H. pylori. The infection always cause a gastric inflammation that may develop into peptic ulcer disease or gastric cancer. Attachment proteins, adhesins, mediate specific adherence of H. pylori to receptor structures on the human gastric mucosa. The best-characterized H. pylori adhesin-receptor interactions are the BabA adhesin and the binding to the fucosylated blood group antigens ABO/Lewis b (Leb) and the SabA adhesin and its binding to the inflammation associated sialyl-Lewis x antigen. During H. pylori infection the availability of receptor structures on the human gastric mucosa changes as a consequence of the host inflammatory and immune responses. Consequently the bacterial population need to adjust its adherence properties to stay colonized. This thesis describes mechanisms that generate H. pylori populations with variable adherence properties and mechanisms for adjustment of adhesin expression levels.In H. pylori strains devoid of Leb-binding, we found bacterial cells with Leb-binding. Isolation of such H. pylori clones demonstrated that the change in receptor binding phenotype was obtained via the mechanisms of homologous recombination and slipped strand mispairing (SSM). Disease presentation in relation to BabA expression was studied in H. pylori infected Mongolian gerbils. We showed that BabA was not essential for colonization but caused severe injury to the gastric mucosa and was turned off during long-term infection by nucleotide changes within the babA gene. Gerbils infected with BabA-weak-expressing strains maintained BabA expressing clones for a longer period than gerbils that were infected with BabA-high-expressing strains. Studies of the gerbil gastric mucosal glycosylation showed that gerbils respond in a similar way as humans and Rhesus monkeys which support gerbils to be a model suitable for studying H. pylori infection and disease outcome in relation to adherence.We studied the SSM mechanism of SabA phase variation and the cognate shift in sLex-binding phenotype and we show sLex-binding activity to be growth phase dependent. H. pylori vesicles were characterized for the major phosholipid and protein components. Virulence factors e.g., VacA, and CagA were identified and both the BabA and the SabA adhesins was shown to be located on the vesicle surface and to mediate specific binding to their cognate receptors present on the human gastric mucosa. H. pylori generate bacterial cells with different receptor binding phenotypes via the mechanisms of homologous recombination, SSM and nucleotide changes. These mechanisms will probably contribute to bacterial fitness by the generation of quasi species populations where some of the clones will be better adapted to the environmental chances during persistent infection.

Introdução e objetivos: Eosinofilia duodenal está associada com parasitoses intestinais e com alergias alimentares, e tem sido sugerida como possível fator etiológico da dispepsia funcional, pela capacidade de causar alterações na motilidade e na sensibilidade do aparelho digestivo. Sua relação com o Helicobacter pylori é pouco conhecida, tendo sido avaliada apenas como achado secundário em alguns estudos, com resultados controversos. Esse estudo tem como objetivos avaliar o papel da infecção gástrica pelo H. pylori no número de eosinófilos duodenais e avaliar a relação dos eosinófilos duodenais com os sintomas da dispepsia funcional. Métodos: foram avaliados 100 pacientes dispépticos funcionais, de acordo com os critérios de Roma III, dos quais 50 foram H. pylori positivos e 50 negativos. Os pacientes foram submetidos à endoscopia digestiva alta com biópsias gástricas e duodenais. A positividade do H. pylori foi avaliada pelo teste de urease e pelo exame histológico (Hematoxilina-eosina e Giemsa). As biópsias duodenais foram avaliadas com hematoxilina-eosina e a número de eosinófilos duodenais foi quantificada pela média de eosinófilos por 5 campos de grande aumento (CGA) aleatórios e não sobrepostos. Eosinofilia duodenal foi definida pela presença de >22 eosinófilos/CGA. As medianas das médias aritméticas dos eosinófilos duodenais por cinco CGA foram comparadas entre os pacientes H. pylori positivos e negativos. Também foi avaliada a relação do número de eosinófilos duodenais com a intensidade e tipo de sintomas dispépticos, determinados por questionário validado (PADYQ). Os eosinófilos duodenais foram avaliados para variáveis demográficas e endoscópicas. Resultados: Pacientes do sexo feminino representaram 88% da amostra e a idade média foi de 41,7 anos As características basais dos pacientes H. pylori positivos e H. pylori negativos foram semelhantes. Apenas um paciente, no grupo H. pylori positivo, apresentou eosinofilia duodenal. As medianas dos eosinófilos duodenais/CGA foram 4,6 [P25-75: 2,8-7,2] nos pacientes H. pylori negativos e 4,7 [P25-75: 3,4-8,4] nos H. pylori positivos (p= 0,403). O número de eosinófilos 8 duodenais foi significativamente maior em pacientes com sintomas mais intensos: pacientes com escore do PADYQ >22 (>50% da pontuação máxima) apresentaram mediana de eosinófilos duodenais/CGA de 5,4 [P25-75: 3,4–7,6] e pacientes com escore ≤22 de 3,4 [P25-75: 2,2–6,0] (p= 0,018). Os pacientes foram divididos em tercis, de acordo com a intensidade dos sintomas: grupo 1 com 31 pacientes (sintomas leves); grupo 2 com 30 pacientes (sintomas moderados); e grupo 3 com 31 pacientes (sintomas acentuados). A mediana dos eosinófilos duodenais/CGA no grupo 1 foi de 3,4 [P25-75: 2,2 -6,0]; no grupo 2 de 4,7 [P25-75: 3,2-6,4]; e o grupo 3 de 5,8 [P25-75: 3,6-8,2] (P=0,033). Houve diferença estatisticamente significativa no número de eosinófilos duodenais entre fumantes e não fumantes (p= 0,030) e entre pacientes com índice de massa corporal (IMC) <25 kg/m2 e IMC ≥ 25 kg/m2 (p= 0,035). Na análise multivariada por regressão linear, os fatores que tiveram influência sobre o número de eosinófilos duodenais foram o tabagismo (p= 0,026) e a intensidade dos sintomas dispépticos (p= 0,039). Conclusões: Esse estudo não mostrou associação entre a infecção pelo H. pylori e a contagem de eosinófilos duodenais, nessa população de pacientes dispépticos funcionais. Entretanto, foi demonstrada uma relação diretamente proporcional e estatisticamente significativa entre o número de eosinófilos duodenais e a intensidade dos sintomas dispépticos. / Background and Aims: Duodenal eosinophilia is associated with intestinal parasitosis and food allergies. It has also been implicated as a potential factor on the etiology of functional dyspepsia, probably by causing changes in digestive tract motility and sensitivity. The association with Helicobacter pylori is poorly understood, and has been only evaluated as a secondary finding in 9 previous studies, with conflicting results. This study aims to evaluate the potential role of gastric H. pylori infection in the duodenal eosinophil count, and the influence of duodenal eosinophils on symptoms in functional dyspeptic subjects. Methods: One hundred functional dyspeptic subjects, according to Rome III criteria, were evaluated, and 50 were H. pylori positive and 50 H. pylori negative. Patients were submitted to upper gastrointestinal endoscopy with gastric and duodenal biopsies. H. pylori positivity was evaluated by urease test and gastric histology (Hematoxylin-eosin and Giemsa). Duodenal biopsies were evaluated with Hematoxylin-Eosin staining, and the duodenal eosinophil count was determined by the mean of eosinophil by 5 random nonoverlapping high power fields (HPF). Duodenal eosinophilia was defined as >22 eosinophils/HPF. The median of the arithmetic means of the duodenal eosinophils counts per high power field were compared between H. pylori positive and H. pylori negative subjects. The relationship between the number of duodenal eosinophils and the intensity and type of dyspeptic symptoms was determined by validated questionnaire (PADYQ). Duodenal eosinophils counts were also evaluated by demographic variables and endoscopic findings. Results: 88% of the subjects were female and the mean age was 41.7 years. Baseline characteristics were similar between H. pylori positive and H. pylori negative subjects. Only one patient, in the H. pylori positive group, had duodenal eosinophilia. The median duodenal eosinophils/HPF were 4.6 [Percentiles 25-75(P25-75): 2.8-7.2] in H. pylori negative and 4.7 [P25-75: 3.4-8.4] in H. pylori positive subjects (p= 0.403). The duodenal eosinophil count was greater in subjects with higher symptoms severity: patients with PADYQ score more than 22 (>50% of the maximum score) had median duodenal eosinophil/HPF of 5.4 [P25-75: 3,4–7,6] and subjects with PADYQ score ≤22 of 3.4 [P25-75: 2.2–6.0] (p= 0.018). The patients were divided into terciles, according to symptoms severity: group 1 with 31 subjects (mild symptoms); group 2 with 30 subjects (moderate symptoms); and group 3 with 31 subjects (severe symptoms). 10 The median duodenal eosinophils/HPF was 3.4 [P25-75: 2.2-6.0] in group 1; 4.7 [P25-75: 3.2-6.4] in group 2; and 5.8 [P25-75: 3.6-8.2] in group 3 (p=0.033). There was a higher duodenal eosinophils count in smokers (current or former) (p=0.030), and subjects with BMI ≥ 25 kg/m2 (p=0.035). In the multivariate analysis by linear regression, the duodenal eosinophil count were influenced by smoking (p = 0.026) and dyspeptic symptoms severity (p= 0.039). Conclusion: This study did not show an association between H. pylori infection and the number of duodenal eosinophils, in this population of functional dyspeptic patients. However, a directly proportional and statistically significant relationship between the number of duodenal eosinophils and the intensity of dyspeptic symptoms has been demonstrated.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O cÃncer gÃstrico apresenta, mundialmente, uma elevada taxa de mortalidade, com alta incidÃncia no Brasil, sendo a infecÃÃo com Helicobacter pylori um fator de risco bem estabelecido. Dependendo da presenÃa de genes de virulÃncia como cagA, cagE, vacA e virB11, H. pylori pode causar respostas inflamatÃrias diferenciadas, apresentando grande quantidade de Ãxido nÃtrico (ON) gerado principalmente por iNOS. Quantidade elevada de ON resulta em acÃmulo de espÃcies reativas do oxigÃnio cuja instabilidade causa danos oxidativos no DNA. A integridade genÃmica à garantida por enzimas de reparo importantes como: APE-1, OGG-1, e PARP-1. Polimorfismos genÃticos que modifiquem a atividade dessas enzimas podem influenciar a capacidade de reparo e, portanto, a susceptibilidade do hospedeiro ao desenvolvimento do cÃncer gÃstrico associado à H. pylori. Assim o objetivo deste estudo foi avaliar a associaÃÃo dos polimorfismos C150T em iNOS, T2197G em APE-1, C1245G em OGG-1 e A40676G em PARP-1 com o genÃtipo de H. pylori em 109 amostra de pacientes diagnosticados com adenocarcinomas gÃstricos atendidos em hospitais de Fortaleza, CearÃ. A identificaÃÃo dos polimorfismos foi feita por PCR-RFLP e a detecÃÃo e genotipagem de H. pylori foram feitas por PCR. Os polimorfismos estudados apresentaram as seguintes freqÃÃncias: iNOS - 78% CC, 21,1% CT e 0,9% TT; PARP-1- 69,7% AA, 26,6% AG e 3,7% GG, para OGG-1 56% CC, 39,4% CG, e 4,6% GG e para APE-1 38,5%TT, 47,7%TG e 13,8% GG. Salienta-se a baixa freqÃÃncia do genÃtipo polimÃrfico (TT) de iNOS e alta frequÃncia do heterozigoto (TG) de APE. Os alelos variantes de iNOS e de PARP-1 foram correlacionadas com indivÃduos &#8804;55 anos, sugerindo que estes polimorfismos estariam associados ao desenvolvimento precoce da neoplasia. Os tumores intestinais localizados na regiÃo nÃo-antro correlacionaram-se com o genÃtipo OGG-1 CG; enquanto que os difusos, localizados no corpo com o genÃtipo AA de PARP-1. H. pylori foi detectada em 92,6% dos casos. Os genes cagA, cagE e virB11 foram detectados em 65,3%, 50,4% e 60,3% dos casos, respectivamente e vacAs1m1 detectado em 72,2%. Os casos foram agrupados considerando os alelos de vacA e a integridade da ilha de patogenicidade cag, sendo os grupos A1 e A2, composto por cepas mais patogÃnicas, o qual foi observado em 33,6% e 13,8% dos pacientes, respectivamente. Na anÃlise individual de cada enzima, observou-se que os indivÃduos portadores dos alelos variantes de APE-1 (TG+GG) estavam infectados com cepas pouco patogÃnicas (p=0,0422). Essas cepas pouco patogÃnicas tambÃm foram associadas aos pacientes portadores do genÃtipo selvagem (AA) de PARP-1 (p=0,0396). Esses dados foram confirmados quando os pacientes infectados por cepas mais virulentas foram comparadas aos infectados por cepas menos virulentas (p=0,046). Analisando apenas o grupo A1 observou-se tambÃm uma correlaÃÃo de APE-1 (TG) com OGG-1(CC). Quando os genÃtipos foram combinados considerando somente as enzimas de reparo estudadas ou duas a duas, verificou-se que parte dos pacientes infectados com o genÃtipo selvagem de PARP-1 eram portadores do alelo variante para pelo menos uma das enzimas e que parte dos pacientes infectados com cepas menos patogÃnicas possuÃam o alelo polimÃrfico de APE-1, independente do genÃtipo da enzima de reparo associada. Somados, esses dados indicam a relevÃncia do polimorfismo da APE-1 no desenvolvimento do cÃncer gÃstrico em indivÃduos infectados com cepas menos virulentas e corroboram com a importÃncia do genÃtipo bacteriano, uma vez que, de maneira geral, indivÃduos com genÃtipo selvagem para as enzimas de reparo estudadas desenvolveram cÃncer gÃstrico quando infectados por cepas virulentas. / Gastric cancer is the most deadly malignant neoplasia worldwide, with high incidence in Brazil and Helicobacter pylori infection is a well-established risk factor. Depending on the presence of virulence genes such as cagA, cagE, vacA and virB11, H. pylori can cause differentiated inflammatory responses, with large amounts of nitric oxide (NO) generated primarily by iNOS. High amount of NO resulting in accumulation of reactive oxygen species can cause DNA oxidative damage. The genomic integrity is guaranteed by important repair enzymes as: APE-1, OGG-1 and PARP-1. Genetic polymorphisms that modify the activity of these enzymes may influence the ability to repair and therefore the host susceptibility to the development of gastric cancer H.pylori associated. Therefore, the goal of this study was to evaluate the association of the C150T polymorphism in iNOS, T2197G in APE-1, C1245G in OGG -1 and A40676G in PARP-1 with H.pylori genotype in 109 cases of patients with gastric adenocarcinoma from hospitals in Fortaleza, CearÃ. The identification of polymorphisms was performed by PCR-RFLP and the detection and genotyping of H.pylori were performed by PCR. The studied polymorphisms showed the following frequencies: iNOS 78% CC, 21.1% CT and 0.9% TT; PARP-1 69.7% AA 26.6% AG and 3.7% GG to OGG -1 56% CC, 39.4% CG and 4.6% GG and APE-1 38.5% TT, 47.7% TG and 13.8% GG. Valuable to note the low frequency of the homozygous polymorphic (TT) of iNOS and the high frequency of heterozygous (TG) from APE-1. The variant alleles of iNOS and PARP-1 were correlated with subjects &#8804; 55 years, suggesting that these polymorphisms were associated with early development of the neoplasia. Intestinal tumors located in the non-antrum were correlated with heterozygous genotype of OGG-1 (CG), while diffuse, located on the body with the AA genotype of PARP-1. H. pylori was detected in 92.6% of cases. The genes cagA, cagE and virB11 were detected in 65.3%, 50.4% and 60.3% of cases respectively and vacAs1m1 was detected in 72.2%. The cases were also grouped considering the alleles of vacA and the integrity of the cag-pathogenicity island. Thus, the groups A1 and A2, consist of more pathogenic strains, were observed in 33.6% and 13.8% of patients, respectively. In the individual analysis of each enzyme, we observed that individuals carrying the variant alleles of APE-1 (TG+GG) were infected with low pathogenic strains (p=0.0422). These low pathogenic strains were also associated with patients carrying the wild genotype (AA) of PARP-1 (p=0.0396). These data were confirmed when patients infected with more virulent strains were compared to those infected with less virulent strains (p = 0.046). Analyzing only the group A1, it was also observed a correlation of APE-1 (TG) with OGG-1 (CC). When genotypes were combined by considering only the repair enzymes studied or two by two, it was found that most patients infected with the wild-type of PARP-1 were carriers of the variant allele for at least one of the enzymes and that most patients infected with less pathogenic strains possess a polymorphic allele of APE-1, independent of the genotype associated with the repair enzyme. Taken together, these data indicate the relevance of the APE-1 polymorphism in the development of gastric cancer in individuals infected with less virulent strains and corroborate the importance of the bacterial genotype, since; in general, individuals with wild-type for enzymes repair studied developed gastric cancer when infected with virulent strains.

Being among the most prevalent of persistent infectious agents in humans worldwide, Helicobacter pylori induces chronic inflammation (gastritis), which may progress to peptic ulceration and stomach cancer. The ability to adhere to the gastric mucosa is considered to be both a colonization and virulence property of H. pylori. For adherence, H. pylori expresses surface-located attachment proteins (adhesins) that bind to specific receptors in the gastric mucosa. The best characterized H. pylori adhesin-receptor interaction is that between the blood group antigen binding adhesin (BabA) and the fucosylated blood group antigens, which are glycans highly expressed in the gastric mucosa. Our recent results have changed the view of the blood group antigen-specific binding mode of H. pylori. We have tested clinical isolates of H. pylori from human populations worldwide for their ability to bind to ABO blood group antigens. The results revealed that more than 95% of isolates from Sweden, Germany, Spain, Japan and Alaska that bind fucosylated blood group antigens, bind both the Lewis b antigen (Leb) (of blood group O) and the blood group A-related antigen A-Lewis b, i.e. they exhibit a generalist type of binding mode. In contrast, the majority of strains (62%) from South American Amerindians bound best to Leb, i.e. they exhibit a specialist blood group “O antigen” binding mode. This specialization in binding coincides with the unique predominance of blood group O in the South American Amerindian populations. Furthermore, we also showed that H. pylori could switch from specialist to generalist binding modes by chromosomal integration of foreign babA gene fragments. A mutant strain lacking the babA gene turned out to adhere to inflamed gastric epithelium, despite the fact that it did not bind Leb. We identified the receptor to which the mutant binds to as the sialyl-dimeric-Lewis x antigen (sdiLex) and found its expression to be associated with persistent H. pylori infection and chronic inflammation, both in humans and Rhesus monkeys. The cognate sialic acid binding adhesin (SabA) was identified by our ReTagging technique. Deletion of sabA caused loss of H. pylori binding to sialylated glycans, and screening of single colony isolates revealed a high frequency of spontaneous on⇒off phase variation in sLex binding. Using erythrocytes as a model for sialyl dependent cell adhesion, we could show that SabA is the sought-after H. pylori sialyl-dependent hemagglutinin. Swedish clinical H. pylori isolates were analyzed for sialyl-dependent hemagglutination (sia-HA), and the sia-HA titers were found to be highly correlated to the levels of sLex binding. Clinical isolates were shown to exhibit several distinct binding modes for sialylated glycans, which suggest that SabA exhibit polymorphism in binding. We also found that SabA binds to sialylated glycans on neutrophil surfaces by mechanisms involving “selectin mimicry”, and that SabA plays an important role in nonopsonic activation of neutrophils. In the human stomach, H. pylori is exposed to selective pressures such as immune and inflammatory responses, and this is reflected by changes in mucosal glycosylation patterns. The high mutation and recombination rates of H. pylori in combination with bio selection will continuously generate clones that are adapted to changes in individual gastric mucosa. Such adaptive selection contributes to the remarkable diversity in binding modes and to the extraordinary chronicity of H. pylori infections worldwide.

The innate immune system is important in both acute and chronic infection. In this thesis, I investigated the effect of H. pylori infection on 1) DCs, key orchestrators of the immune system, and 2) autophagy, recently identified as an important component of innate immunity. I determined that H. pylori activates the STAT3 pathway in DCs, increasing DC maturation and inducing production of IL-10, IL-12p40 and TNF-α, without IL-12p70. This cytokine profile may favour an immunoregulatory response, promoting persistent H. pylori infection. In addition I determined that H. pylori’s VacA toxin induced autophagy, ROS production and Parkin aggregation which has been implicated in mediating autophagy in response to mitochondrial damage. Thus H. pylori alters these key effectors of innate immunity which may play a role in promoting its chronic infection and disease.

The innate immune system is important in both acute and chronic infection. In this thesis, I investigated the effect of H. pylori infection on 1) DCs, key orchestrators of the immune system, and 2) autophagy, recently identified as an important component of innate immunity. I determined that H. pylori activates the STAT3 pathway in DCs, increasing DC maturation and inducing production of IL-10, IL-12p40 and TNF-α, without IL-12p70. This cytokine profile may favour an immunoregulatory response, promoting persistent H. pylori infection. In addition I determined that H. pylori’s VacA toxin induced autophagy, ROS production and Parkin aggregation which has been implicated in mediating autophagy in response to mitochondrial damage. Thus H. pylori alters these key effectors of innate immunity which may play a role in promoting its chronic infection and disease.

Bacterial cells were once treated as membrane-enclosed bags of cytoplasm: a homogeneous, undifferentiated suspension in which polymers (proteins, nucleic acids, etc.) and small molecules diffused freely to interact with each other. Biochemical studies have determined the molecular mechanisms underlying the biological processes of metabolism, replication and transcription-translation, etc. However, recent advancements in optical techniques armed with fluorescent tags for proteins and nucleic acids have increased our ability to peer into the interior of live bacterial cells. This has revealed an organized layout of multi-protein complexes, or molecular machines, dedicated to specific functions at defined sub-cellular locations; the timing of their assembly and/or rates of their activity being determined by available nutrition and environmental signals from the niche occupied by the organism. In the present study, we have attempted to identify the intracellular location and organization of the molecular machines assembled for protein synthesis (ribosomes), DNA replication (replisomes) and cell division (divisome) in different bacteria. We have used the model system Escherichia coli as well as Helicobacter pylori and mycobacterial strains (Mycobacterium marinum and Mycobacterium smegmatis), which grow at different rates and move to dormancy late into stationary phase Bacterial nucleoid plays a major role in organizing the location and movement of active ribosomes, replisomes and placement of divisome. While the active ribosomes appear to follow the dynamic folds of the bacterial nucleoid during cell growth in E. coli, inactive ribosomes appear to accumulate near the periphery. The replisome in H. pylori was visualized as a sharp, single focus upon SSB and DnaB co-localization in growing helical rods but disassembled into diffused fluorescence when the cells attained non-replicative coccoid stage. Our investigation into mycobacterial life-cycle revealed unique features such as an absence of a dedicated mid-cell site for divisome assembly and endosporulation upon entry into stationary phase. In brief, we present the cell cycle-dependent subcellular organization of molecular machines in bacteria.

Introdução e objetivos: Eosinofilia duodenal está associada com parasitoses intestinais e com alergias alimentares, e tem sido sugerida como possível fator etiológico da dispepsia funcional, pela capacidade de causar alterações na motilidade e na sensibilidade do aparelho digestivo. Sua relação com o Helicobacter pylori é pouco conhecida, tendo sido avaliada apenas como achado secundário em alguns estudos, com resultados controversos. Esse estudo tem como objetivos avaliar o papel da infecção gástrica pelo H. pylori no número de eosinófilos duodenais e avaliar a relação dos eosinófilos duodenais com os sintomas da dispepsia funcional. Métodos: foram avaliados 100 pacientes dispépticos funcionais, de acordo com os critérios de Roma III, dos quais 50 foram H. pylori positivos e 50 negativos. Os pacientes foram submetidos à endoscopia digestiva alta com biópsias gástricas e duodenais. A positividade do H. pylori foi avaliada pelo teste de urease e pelo exame histológico (Hematoxilina-eosina e Giemsa). As biópsias duodenais foram avaliadas com hematoxilina-eosina e a número de eosinófilos duodenais foi quantificada pela média de eosinófilos por 5 campos de grande aumento (CGA) aleatórios e não sobrepostos. Eosinofilia duodenal foi definida pela presença de >22 eosinófilos/CGA. As medianas das médias aritméticas dos eosinófilos duodenais por cinco CGA foram comparadas entre os pacientes H. pylori positivos e negativos. Também foi avaliada a relação do número de eosinófilos duodenais com a intensidade e tipo de sintomas dispépticos, determinados por questionário validado (PADYQ). Os eosinófilos duodenais foram avaliados para variáveis demográficas e endoscópicas. Resultados: Pacientes do sexo feminino representaram 88% da amostra e a idade média foi de 41,7 anos As características basais dos pacientes H. pylori positivos e H. pylori negativos foram semelhantes. Apenas um paciente, no grupo H. pylori positivo, apresentou eosinofilia duodenal. As medianas dos eosinófilos duodenais/CGA foram 4,6 [P25-75: 2,8-7,2] nos pacientes H. pylori negativos e 4,7 [P25-75: 3,4-8,4] nos H. pylori positivos (p= 0,403). O número de eosinófilos 8 duodenais foi significativamente maior em pacientes com sintomas mais intensos: pacientes com escore do PADYQ >22 (>50% da pontuação máxima) apresentaram mediana de eosinófilos duodenais/CGA de 5,4 [P25-75: 3,4–7,6] e pacientes com escore ≤22 de 3,4 [P25-75: 2,2–6,0] (p= 0,018). Os pacientes foram divididos em tercis, de acordo com a intensidade dos sintomas: grupo 1 com 31 pacientes (sintomas leves); grupo 2 com 30 pacientes (sintomas moderados); e grupo 3 com 31 pacientes (sintomas acentuados). A mediana dos eosinófilos duodenais/CGA no grupo 1 foi de 3,4 [P25-75: 2,2 -6,0]; no grupo 2 de 4,7 [P25-75: 3,2-6,4]; e o grupo 3 de 5,8 [P25-75: 3,6-8,2] (P=0,033). Houve diferença estatisticamente significativa no número de eosinófilos duodenais entre fumantes e não fumantes (p= 0,030) e entre pacientes com índice de massa corporal (IMC) <25 kg/m2 e IMC ≥ 25 kg/m2 (p= 0,035). Na análise multivariada por regressão linear, os fatores que tiveram influência sobre o número de eosinófilos duodenais foram o tabagismo (p= 0,026) e a intensidade dos sintomas dispépticos (p= 0,039). Conclusões: Esse estudo não mostrou associação entre a infecção pelo H. pylori e a contagem de eosinófilos duodenais, nessa população de pacientes dispépticos funcionais. Entretanto, foi demonstrada uma relação diretamente proporcional e estatisticamente significativa entre o número de eosinófilos duodenais e a intensidade dos sintomas dispépticos. / Background and Aims: Duodenal eosinophilia is associated with intestinal parasitosis and food allergies. It has also been implicated as a potential factor on the etiology of functional dyspepsia, probably by causing changes in digestive tract motility and sensitivity. The association with Helicobacter pylori is poorly understood, and has been only evaluated as a secondary finding in 9 previous studies, with conflicting results. This study aims to evaluate the potential role of gastric H. pylori infection in the duodenal eosinophil count, and the influence of duodenal eosinophils on symptoms in functional dyspeptic subjects. Methods: One hundred functional dyspeptic subjects, according to Rome III criteria, were evaluated, and 50 were H. pylori positive and 50 H. pylori negative. Patients were submitted to upper gastrointestinal endoscopy with gastric and duodenal biopsies. H. pylori positivity was evaluated by urease test and gastric histology (Hematoxylin-eosin and Giemsa). Duodenal biopsies were evaluated with Hematoxylin-Eosin staining, and the duodenal eosinophil count was determined by the mean of eosinophil by 5 random nonoverlapping high power fields (HPF). Duodenal eosinophilia was defined as >22 eosinophils/HPF. The median of the arithmetic means of the duodenal eosinophils counts per high power field were compared between H. pylori positive and H. pylori negative subjects. The relationship between the number of duodenal eosinophils and the intensity and type of dyspeptic symptoms was determined by validated questionnaire (PADYQ). Duodenal eosinophils counts were also evaluated by demographic variables and endoscopic findings. Results: 88% of the subjects were female and the mean age was 41.7 years. Baseline characteristics were similar between H. pylori positive and H. pylori negative subjects. Only one patient, in the H. pylori positive group, had duodenal eosinophilia. The median duodenal eosinophils/HPF were 4.6 [Percentiles 25-75(P25-75): 2.8-7.2] in H. pylori negative and 4.7 [P25-75: 3.4-8.4] in H. pylori positive subjects (p= 0.403). The duodenal eosinophil count was greater in subjects with higher symptoms severity: patients with PADYQ score more than 22 (>50% of the maximum score) had median duodenal eosinophil/HPF of 5.4 [P25-75: 3,4–7,6] and subjects with PADYQ score ≤22 of 3.4 [P25-75: 2.2–6.0] (p= 0.018). The patients were divided into terciles, according to symptoms severity: group 1 with 31 subjects (mild symptoms); group 2 with 30 subjects (moderate symptoms); and group 3 with 31 subjects (severe symptoms). 10 The median duodenal eosinophils/HPF was 3.4 [P25-75: 2.2-6.0] in group 1; 4.7 [P25-75: 3.2-6.4] in group 2; and 5.8 [P25-75: 3.6-8.2] in group 3 (p=0.033). There was a higher duodenal eosinophils count in smokers (current or former) (p=0.030), and subjects with BMI ≥ 25 kg/m2 (p=0.035). In the multivariate analysis by linear regression, the duodenal eosinophil count were influenced by smoking (p = 0.026) and dyspeptic symptoms severity (p= 0.039). Conclusion: This study did not show an association between H. pylori infection and the number of duodenal eosinophils, in this population of functional dyspeptic patients. However, a directly proportional and statistically significant relationship between the number of duodenal eosinophils and the intensity of dyspeptic symptoms has been demonstrated.

Introduction: Helicobater pylori interferes with vitamin C homeostasis and low levels of vitamin C may be a risk factor for H. pylori infection. Objective: to investigate the vitamin C serum and gastric juice levels of patients infected and non-infected by H. pylori. Additionally assess the association of vitamin C levels with H.pylori status, gastric pH and histological changes of gastric mucosa of dyspeptic patients with normal upper GI endoscopy. Methods: We studied 42 dyspeptic patients undergoing upper GI endoscopy had biopsies for diagnosis of H. pylori infection and gastric mucosa changes. We also collected 5 ml of blood (for dosage of serum vitamin C) and 10 ml of gastric juice. We measure pH of gastric juice. We used high-performance liquid chromatography to assess Vitamin C in serum and gastric juice. Results: The average age was 52 years (SD±11.8), 84.4% female, overweight BMI median 27,7) with adequate dietary intake of vitamin C. Twenty four (53.3%) patients had H. pylori infection. The median serum vitamin C levels in infected and non-infected was 3.9 and 3.4 μg (p = 0.59). The median gastric juice levels of vitamin C was 9,8 μg in infected and 18.4 μg in non-infected (p = 0.03). The histology showed normal mucosa in eight 8 (20%), chronic non-atrophic gastritis in 23 (55%) and chronic atrophic gastritis in 11 (26%) patients. There was no significant association of histology with Vitamin C serum levels (p = 0.26) or gastric (p = 0.29). Conclusion: Vitamin C serum levels were within the normal range, and most patients had adequate food ingestion of vitamin C. Serum levels of vitamin C were similar in individuals infected and non-infected while vitamin C levels in the gastric juice of patients infected were reduced compared with patients non-infected. Serum levels of vitamin C were higher in the gastric juice than in serum, regardless the patients were infected or not. / Introdução: A infecção por Helicobater pylori influencia na homeostase da vitamina C e níveis baixos da mesma podem determinar risco para a infecção. Objetivo: Investigar os níveis de vitamina C no soro e suco gástrico de pacientes infectados e não infectados por H. pylori e estudar sua associação com a infecção, com o pH gástrico e com as alterações anatomopatológicas da mucosa gástrica de pacientes dispépticos funcionais. Métodos: Foram estudados 42 pacientes dispépticos submetidos a endoscopia digestiva alta e biópsia para diagnóstico da infecção por H. pylori e de alterações anatomopatológicas da mucosa gástrica, classificadas de acordo com o Sistema Sidney. Aspirado suco gástrico com mensuração do pH e coleta de 5 ml de sangue para dosagem de vitamina C no soro. A dosagem da Vitamina C no soro e no suco gástrico foi realizada por cromatografia líquida de alta performance. Resultados: A idade média foi de 52 anos (DP±11,8), 84,4% do sexo feminino, com sobrepeso (IMC mediana=27,7), com adequada ingestão alimentar de vitamina C. Vinte e quatro (53,3%) pacientes apresentavam infecção por H. pylori. A mediana dos níveis de vitamina C no soro dos infectados foi 3,9 μg/ml e nos não infectados 3,4 μg/ml (p=0,59). A mediana dos níveis de vitamina C no suco gástrico foi 9,8 μg/ml nos infectados e 18,4 μg/ml nos não infectados (p=0,03). A histologia demonstrou mucosa normal em 8 (20%), gastrite crônica não atrófica em 23 (55%) e gastrite crônica atrófica em 11 (26%) pacientes, sem associação significativa com os níveis séricos (p=0,26) ou gástricos (p=0,29) da vitamina C. Conclusões: O nível sérico da vitamina C nos pacientes estudados esteve dentro dos valores de referência normais e o consumo de vitamina C foi adequado na maioria. Os níveis séricos da vitamina C foram semelhantes em indivíduos infectados e não infectados enquanto os níveis de vitamina C apresentaram-se reduzidos no suco gástrico dos pacientes infectados por H. pylori em comparação com os não infectados. Os níveis séricos de vitamina C foram mais altos no suco gástrico do que no soro, independentemente da condição de infectado ou não.