Search

Aims: To understand whether the variability found in the proteome of Helicobacter pylori relates to the genomic methylation, virulence and associated gastric disease. Methods and Results: We applied the Minimum-Common-Restriction- Modification (MCRM) algorithm to genomic methylation data of 30 Portuguese H. pylori strains, obtained by genome sensitivity to Type II restriction enzymes’ digestion. All the generated dendrograms presented three clusters with no association with gastric disease. Comparative analysis of twodimensional gel electrophoresis (2DE) maps obtained for total protein extracts of 10 of these strains, representative of the three main clusters, revealed that among 70 matched protein spots (in a universe of 300), 16 were differently abundant (P < 0 05) among clusters. Of these, 13 proteins appear to be related to the cagA genotype or gastric disease. The abundance of three protein species, DnaK, GlnA and HylB, appeared to be dictated by the methylation status of their gene promoter. Conclusions: Variations in the proteome profile of strains with common geographic origin appear to be related to differences in cagA genotype or gastric disease, rather than to clusters organized according to strain genomic methylation. Significance and Impact of the Study: The simultaneous study of the genomic methylation and proteome is important to correlate epigenetic modifications with gene expression and pathogen virulence. FCT

Helicobacter pylori infection is the major cause of paediatric peptic ulcer disease (PUD). In children with no other aetiology for the disease, this rare event occurs shortly after infection, presuming a still poorly understood higher susceptibility of the patient and highlighting the virulence of the implicated strain. Recently, we showed that the enhanced virulence of a group of paediatric ulcerogenic-strains result from a synergy between their ability to better adapt to the hostility of their niche and the expression of cagA, vacAs1, oipA ‘‘on’’ status, homB and jhp5621. Accordingly, these ulcerogenic strains share a particular proteome profile, providing them with better antioxidant defences, a metabolism favouring the biosynthesis of aromatic amino acids and higher motility1. Corroborating these findings, our preliminary data on electronic microscopic analyses demonstrated the presence of more abundant flagella in PUD-associated paediatric strains, in contrast to the control strain, a paediatric strain associated with non-ulcer dyspepsia (NUD). Compared with paediatric NUD-associated isolates, ulcerogenic H. pylori strains present a greater ability to induce a marked decrease in the gastric cells’ viability and to cause them severe cytoskeleton damage and mucins’ production/secretion impairment1. To uncover the underlying molecular mechanisms, we are now characterizing the modifications induced by these strains in the proteome of human gastric cells, during in vitro infection, by two-dimensional gel electrophoresis followed by mass-spectrometry. Research Grant 2011 – Sociedade Portuguesa de Gastrenterologia.

Until recently, Helicobacter pylori was considered a bacterium without prophages. The presence of an incomplete prophage sequence in strain B38 and a complete prophage sequence in strain B45 showed otherwise. Using a PCR strategy, based on degenerated primers designed after aligning bacteriophage integrase genes from H. pylori strains B38 and B45, and H. acinonychis prophage II, we found that integrase sequence was present in 21.4% (73/341) of the H. pylori clinical strains tested. The phylogenetic analysis of the sequenced region revealed that strains cluster according to their geographic origin, but not to their pathology. We have applied the same methodology to additional 147 European strains and 77 African strains, determining the presence of integrase sequence in 25.2% (37/147) of the former and in 19.5% (15/77) of the latter. Currently, we have a total of 565 strains screened for the presence of integrase gene, with 125 positive for this sequence (22.1%). To understand if these integrase sequences belong to reminiscent or complete prophages we are also screening for the presence of other prophage coding sequences. Among integrase positive strains, we found 19.2% (5/26) positive strains for the primase sequence and 53.3% (8/15) for the presence of the end of the phage. Presently, we are running the sequencing of the PCR amplified products in order to conduct the phylogenetic analysis. The results reinforce the abundance of prophages sequences in H. pylori and suggest that the majority of them belong to reminiscent prophages integrated within the bacterium genome.

Genomic-methylation typing method, based on strains’ Restriction/Modification systems, confirmed the genetic variability of Helicobacter pylori. According to this, strains isolated from patients of the same family, or from the same geographic region, cluster together. The analysis of proteome’s variability of these clusters has been a missing topic. We applied the Minimum-Common-Restriction-Modification (MCRM) algorithm to genomic-methylation data of 30 H. pylori strains, isolated from Portuguese patients, presenting different gastric diseases. 100% of generated dendrograms presented three incipient clusters (C1, C2 and C3), which is characteristic of strains sharing the geographic origin. The same pattern was observed when the MCRM algorithm was applied to a subset of strains (2 of C1, 2 of C2, 4 of C3 and two outsiders). These were heterogeneous regarding their cagA and vacA genotypes and in terms of patient’s age, gender and gastric disease. Comparative analysis of two-dimensional-gel-electrophoresis (2-DE) maps, obtained for total-protein extracts of each strain, revealed that among 70 matched protein spots (in a universe of 300), 16 were differently abundant (p < .05) among clusters. These proteins’ abundance was then compared having the 2DE-maps regrouped according to the strain’s cagA-genotype or its association with gastric disease. We concluded that abundance variations of at least 12 proteins were dictated by differences in virulence, rather than cluster proximity. Therefore, although the genome-methylation typing method discriminates differences in restriction/modification enzymes, strains of each generated cluster do not share a marked particular proteome, arguing that strains with common geographic origin vary greatly in virulence.

Background: Helicobacter pylori colonizes the human stomach and is associated with gastritis, peptic ulcer, and gastric cancer. This ubiquitous association between H. pylori and humans is thought to be present since the origin of modern humans. The H. pylori genome encodes for an exceptional number of restriction and modifications (R-M) systems. To evaluate if R-M systems are an adequate tool to determine the geographic distribution of H. pylori strains, we typed 221 strains from Africa, America, Asia, and Europe, and evaluated the expression of different 29 methyltransferases. Results: Independence tests and logistic regression models revealed that ten R-M systems correlate with geographical localization. The distribution pattern of these methyltransferases may have been originated by co-divergence of regional H. pylori after its human host migrated out of Africa. The expression of specific methyltransferases in the H. pylori population may also reflect the genetic and cultural background of its human host. Methyltransferases common to all strains, M. HhaI and M. NaeI, are likely conserved in H. pylori, and may have been present in the bacteria genome since the human diaspora out of Africa. Conclusion: This study indicates that some methyltransferases are useful geomarkers, which allow discrimination of bacterial populations, and that can be added to our tools to investigate human migrations.. - New England Biolabs, Inc. (USA). - We thank Lurdes Monteiro and Sebastian Suerbaum for the H. pylori strains, Patricia Fonseca and Rui Moreira for critical review of the manuscript, and Afonso Cavaco, Antonio Belo and Dinis Pestana for helping on the logistic regression analysis. This work was partially supported by New England Biolabs, Inc. (USA).

The genome sequences of three Helicobacter pylori strains revealed an abundant number of putative restriction and modification (R-M) systems within a small genome (1.60 to 1.67 Mb). Each R-M system includes an endonuclease that cleaves a specific DNA sequence and a DNA methyltransferase that methylates either adenosine or cytosine within the same DNA sequence. These are believed to be a defense mechanism, protecting bacteria from foreign DNA. They have been classified as selfish genetic elements; in some instances it has been shown that they are not easily lost from their host cell. Possibly because of this phenomenon, the H. pylori genome is very rich in R-M systems, with considerable variation in potential recognition sequences. For this reason the protective aspect of the methyltransferase gene has been proposed as a tool for typing H. pylori isolates. We studied the expression of H. pylori methyltransferases by digesting the genomic DNAs of 50 strains with 31 restriction endonucleases. We conclude that methyltransferase diversity is sufficiently high to enable the use of the genomic methylation status as a typing tool. The stability of methyltransferase expression was assessed by comparing the methylation status of genomic DNAs from strains that were isolated either from the same patient at different times or from different stomach locations (antrum and corpus). We found a group of five methyltransferases common to all tested strains. These five may be characteristic of the genetic pool analyzed, and their biological role may be important in the host/bacterium interaction.

Helicobacter pylori es una bacteria patógena capaz de infectar el estómago humano. Se considera la infección bacteriana crónica más frecuente en todo el mundo. Todos los individuos infectados con H. pylori presentan gastritis crónica, y el 20% pueden progresar a enfermedades como úlcera péptica, adenocarcinoma o linfoma gástrico tipo MALT. Numerosos factores se han descrito como responsables de la progresión de la infección, dentro de ellos, varios factores de virulencia de H. pylori. Un desafío crítico que presenta la infección por H. pylori, son los altos niveles de resistencia a los antibióticos. Se han informado tasas de efectividad del tratamiento antibiótico tan bajas como del 57%, cuando la mínima aceptable para un tratamiento empírico es del 90%. Además de ser una bacteria virulenta y resistente a los antibióticos, H. pylori es una bacteria genéticamente diversa. Fenómeno que puede estar influenciado entre otras causas, por la presencia de elementos genómicos móviles, como los profagos. Esta diversidad bacteriana se ha asociado con el origen geográfico de las poblaciones y cuando se ha analizado en conjunto con la diversidad de los profagos, la mayoría de las observaciones indican una concordancia filogeográfica; sin embargo, algunos profagos de H. pylori se han asignado a poblaciones distintas de su anfitrión, mientras que otros han exhibido signos de recombinación entre poblaciones. Teniendo en cuenta estos antecedentes, el objetivo de este trabajo fue caracterizar molecularmente genomas colombianos de H. pylori, incluyendo filogenia, resistoma, viruloma y caracterización de profagos. Para ello, se analizaron 221 genomas colombianos de H. pylori, los cuales incluyeron 29 genomas secuenciados en este estudio y 192 genomas recolectados de bases de datos públicas. Para la determinación del viruloma, se realizó la caracterización de los principales factores de virulencia de H. pylori: cagA (motivos EPIYA), cagPAI, vacA (regiones m, s e i), iceA, babA y genes de adherencia. Para conocer el resistoma, se realizó la búsqueda de mutaciones en los genes y/o proteínas relacionadas con resistencia a Claritromicina (CLA), Tetraciclina (TET), Levofloxacina (LEV), Amoxicilina (AMX) y Metronidazol (MTZ). Para el análisis de filogenia, se realizó caracterización por MLST (Multi-locus sequence typing) y análisis mediante SNP (polimorfismos de nucleótido simple), adicionalmente se realizó una caracterización del pangenoma colombiano de H. pylori utilizando el software Roary. La búsqueda y caracterización de profagos, se realizó utilizando los genes marcadores integrasa y holin, así como de la secuencia completa de los profagos. Se realizó una caracterización genómica y filogenética de los profagos colombianos de H. pylori. El análisis de los genomas mostró que estos provenían de aislamientos de H. pylori obtenidos en diferentes regiones de Colombia entre los años 2000 y 2018. Al analizar el viruloma se logró evidenciar que el genotipo de virulencia más frecuentemente encontrado fue vacAs1b i1 m1 / babA2 / iceA2 / cagPAI parcialmente delecionado con cagA+ (EPIYA-ABC), un genotipo definido como de alta virulencia. El análisis de resistoma permitió evidenciar la presencia de mutaciones asociadas a resistencia a CLA en el 3,6% de los genomas analizados, a TET en el 3,61%, a AMX en el 7,23%, a LEV en el 10,26% y a MTZ en el 75,7%. Se encontraron 32 (14,4%) genomas con mutaciones ligadas a resistencia frente a más de un antibiótico. Estos análisis reflejan las características de virulencia y las mutaciones asociadas a resistencia de las cepas que han circulado en el país a lo largo de las dos últimas décadas. El estudio de la estructura poblacional revelo la presencia de un grupo clonal compuesto exclusivamente por aislamientos colombianos. El análisis de pangenoma permitió evidenciar que el pangenoma colombiano de H. pylori es abierto y muy diverso, lo cual está relacionado con la amplia diversidad genética y la historia evolutiva de esta bacteria humana. Los análisis de búsqueda y caracterización de profagos permitieron encontrar profagos o parte de ellos en 11 de los genomas analizados, incluyendo los genomas secuenciados en este estudio y los colombianos disponibles en bases de datos públicas. Los análisis de filogenia para estos profagos mostraron evidencia de una nueva población filogenética formada exclusivamente por profagos de origen colombiano. Este nuevo grupo clonal fue denominado hpColombia. Los resultados obtenidos aquí permiten ampliar el conocimiento de la genómica de aislamientos colombianos de H. pylori, dar a conocer la presencia de profagos en éstos, comprobar la presencia de un grupo clonal específico para los profagos colombianos de H. pylori y también para sus bacterias hospederas. Adicionalmente, los análisis realizados aquí permiten verificar que la genómica comparativa es una herramienta útil en la evaluación de varios elementos que contribuyen a la diversidad entre los genomas de H. pylori. Ministerio de Ciencia y Tecnología de Colombia - MinCiencias- Proyecto:120380763025/2018 Helicobacter pylori is a pathogenic bacterium capable of infecting the human stomach. This infection is considered the most common chronic bacterial infection in the world. All individuals infected with H. pylori develop chronic gastritis, and 20% of them may progress to diseases such as peptic ulcer, adenocarcinoma, or gastric MALT lymphoma. Numerous factors have been described as responsible for the progression of the infection. Among them, several factors of virulence of H. pylori. Another critical challenge presented by H. pylori infection is its high levels of resistance to antibiotics. Antibiotic treatment effectiveness rates as low as 57% have been reported, when the minimum acceptable for empirical treatment is 90%. In addition to being a virulent and antibiotic-resistant bacterium, H. pylori is a genetically diverse bacterium, which can be influenced by the presence of mobile genomic elements, such as prophages. This diversity has been associated with the geographic origin of the populations. Most observations indicate a phylogeographic agreement between prophage and bacterial genes; however, some H. pylori prophages have been assigned to populations other than their host, while others show signs of recombination between populations. Considering these antecedents, the objective of this work was to characterize molecularly Colombian H. pylori genomes. The characterization included phylogeny, resistome, virulome, and the presence of prophages. For this, 221 Colombian H. pylori genomes were analyzed, including 29 genomes sequenced in this study and 192 genomes collected from public databases. To determine the virulome, the H. pylori mayor virulence factors were characterized: cagA (including EPIYA motifs), cagPAI, vacA (m, s and i regions), iceA, babA, and some adherence genes. A search for mutations in genes and/or proteins related to resistance to Clarithromycin (CLA), Tetracycline (TET), Levofloxacin (LEV), Amoxicillin (AMX), and Metronidazole (MTZ) was made to determine the resistome. For the phylogeny analysis, the characterization was performed by MLST (Multi-locus sequence typing) and SNP (single nucleotide polymorphisms); additionally, a description of the Colombian H. pylori pangenome was performed using Roary software. The characterization of prophages was carried out by searching the integrase and holin genes and the genomic and phylogenetic characterization of the complete sequence of the prophages. The genomes analysis showed that they came from H. pylori isolates obtained in different regions of Colombia between 2000 and 2018. Virulome analysis showed that the most frequently found virulence genotype was vacAs1bi1m1 / babA2 / iceA2 / cagPAI partially deleted with cagA+ (EPIYA-ABC), a genotype defined as high virulence. The resistome analysis revealed the presence of mutations associated with resistance to CLA in 3.6% of the genomes analyzed, to TET in 3.61%, to AMX in 7.23%, to LEV in 10, 26%, and MTZ at 75.7%. 32 (14.4%) genomes were found with mutations linked to resistance to more than one antibiotic. These analyzes reflect the virulence characteristics and the resistance-associated mutations present in H. pylori strains that have circulated in the country over the last two decades. The study of the population structure by MLST showed that the Colombian isolates belong to the clonal group hpAfrica1 and mainly to the group HpEurope; the complementary analyzes showed the presence of a clonal group composed exclusively of Colombian isolates. The pangenome analysis showed that the Colombian H. pylori pangenome is open and very diverse. In these analyzes, the presence of some marker genes of geographic distribution was not found. The analysis of prophages allowed us to find three complete prophages and two incomplete prophages in the sequenced isolates and the presence of prophages in six genomes recovered from public databases. Phylogenetic analyzes for these prophages showed evidence of a new phylogenetic population made up exclusively of prophages of Colombian origin. This new clonal group was named hpColombia. The results obtained here allow us to expand the knowledge of the genomics of Colombian H. pylori isolates. Likewise, the results will enable us to inform the presence of prophages in these genomes and verify the existence of a specific clonal group for this region of both prophages and their host bacteria. The analyses carried out here allow us to confirm that comparative genomics is a helpful tool in evaluating various elements that contribute to the diversity among H. pylori genomes. Doctor en Ciencias Biológicas Doctorado https://orcid.org/0000-0002-7451-3189 https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0000619191

Tese de mestrado em Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2021 Submitted by Cristina Manessiez (camanessiez@fc.ul.pt) on 2021-05-10T18:11:26Z No. of bitstreams: 1 ulfc126236_tm_Luis_Tanoeiro.pdf: 3038054 bytes, checksum: 4b9ef634cf0f7556c877020c5cfe5876 (MD5) Made available in DSpace on 2021-05-10T18:11:36Z (GMT). No. of bitstreams: 1 ulfc126236_tm_Luis_Tanoeiro.pdf: 3038054 bytes, checksum: 4b9ef634cf0f7556c877020c5cfe5876 (MD5) Previous issue date: 2021

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2013 Submitted by Lurdes Saramago (lurdes.saramago@fc.ul.pt) on 2013-11-27T18:57:03Z No. of bitstreams: 1 ulfc103126_tm_andreia_timoteo.pdf: 1291466 bytes, checksum: c78c6bb3977d8f9ff10ea463cf577876 (MD5) Made available in DSpace on 2013-11-27T18:57:16Z (GMT). No. of bitstreams: 1 ulfc103126_tm_andreia_timoteo.pdf: 1291466 bytes, checksum: c78c6bb3977d8f9ff10ea463cf577876 (MD5) Previous issue date: 2013