Browsing by Author "Vale, Filipa"
Now showing 1 - 6 of 6
Results Per Page
Sort Options
- Gene content, phage cycle regulation model and prophage inactivation disclosed by prophage genomics in the Helicobacter pylori Genome ProjectPublication . Vale, Filipa; HpGP Research Network; Roberts, Richard; Kobayashi, Ichizo; Camargo, Constanza; Rabkin, Charles; HpGP Research NetworkProphages can have major clinical implications through their ability to change pathogenic bacterial traits. There is limited understanding of the prophage role in ecological, evolutionary, adaptive processes and pathogenicity of Helicobacter pylori, a widespread bacterium causally associated with gastric cancer. Inferring the exact prophage genomic location and completeness requires complete genomes. The international Helicobacter pylori Genome Project (HpGP) dataset comprises 1011 H. pylori complete clinical genomes enriched with epigenetic data. We thoroughly evaluated the H. pylori prophage genomic content in the HpGP dataset. We investigated population evolutionary dynamics through phylogenetic and pangenome analyses. Additionally, we identified genome rearrangements and assessed the impact of prophage presence on bacterial gene disruption and methylome. We found that 29.5% (298) of the HpGP genomes contain prophages, of which only 32.2% (96) were complete, minimizing the burden of prophage carriage. The prevalence of H. pylori prophage sequences was variable by geography and ancestry, but not by disease status of the human host. Prophage insertion occasionally results in gene disruption that can change the global bacterial epigenome. Gene function prediction allowed the development of the first model for lysogenic-lytic cycle regulation in H. pylori. We have disclosed new prophage inactivation mechanisms that appear to occur by genome rearrangement, merger with other mobile elements, and pseudogene accumulation. Our analysis provides a comprehensive framework for H. pylori prophage biological and genomics, offering insights into lysogeny regulation and bacterial adaptation to prophages.
- Insights into macrolide resistance in Arcobacter butzleri: potential resistance mechanisms and impact on bacterial fitness and virulencePublication . Couto, Francisca; Martins, Inês; Vale, Filipa; Domingues, Fernanda; Oleastro, Mónica; Ferreira, SusanaBackground: Macrolides are recommended for treating the emerging enteropathogen Arcobacter butzleri; nonetheless, this bacterium often exhibits highly variable resistance rates, and the mechanisms behind this resistance phenotype remain largely unexplored. Objectives: To understand the phenotypic and genotypic consequences associated with the acquisition of erythromycin resistance in A. butzleri, as well as the effects on the fitness of this species. Methods: Resistant strains resulting from spontaneous mutations and adaptive laboratory evolution under increasing erythromycin concentrations were examined regarding their cross-resistance and collateral susceptibility profiles. Genetic causes of phenotypic antibiotic resistance were analysed by sequencing and bioinformatics, with functional correlation through ethidium bromide accumulation assays. Growth profiles in the presence and absence of erythromycin, motility and biofilm formation abilities were assessed to detect potential changes in fitness and virulence. Results: Clones from spontaneous mutation rate evolution demonstrated decreased susceptibility to erythromycin and other classes of antibiotics, associated with mutations in the transcriptional repressor areR, causing overexpression of the AreABC efflux pump. In turn, WGS analysis of the evolved strain showed additional mutations in the ribosomal proteins L4 and L22 and in the areR gene. Furthermore, the acquisition of macrolide resistance altered A. butzleri virulence and entailed a high biological cost. Conclusions: The findings of this study have proved that efflux activity contributes synergistically with mutations in the ribosomal proteins L4 and L22 to A. butzleri's high-level macrolide resistance. The results further suggest an impact on the bacterial physiology and virulence, with the increased fitness cost justifying the low worldwide prevalence of high-level resistant circulating strains.
- Proteome analysis of clustered helicobacter pylori strainsPublication . Vitoriano, Inês; Vítor, Jorge; Oleastro, Mónica; Roxo-Rosa, Mónica; Vale, FilipaGenomic-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.
- Proteome variability among Helicobacter pylori isolates clustered according to genomic methylationPublication . Vitoriano, Inês; Vítor, Jorge; Oleastro, Mónica; Roxo-Rosa, Mónica; Vale, FilipaAims: 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.
- Screening of Prophage Sequences Among Helicobacter PyloriPublication . Timóteo, Andreia; Breurec, S.; Oleastro, Mónica; Roxo-Rosa, Mónica; Vítor, Jorge; Lehours, Phillipe; Vale, FilipaUntil 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.
- The ulcerogenic profile of Helicobacter pylori paediatric strains associated with peptic ulcer disease.Publication . Vitoriano, Inês; Saraiva-Pava, Kathy; Rocha-Gonçalves, António; Alves-Matos, Pedro; Vale, Filipa; Santos, Andrea; Lopes, Ana Isabel; Oleastro, Mónica; Roxo-Rosa, MónicaHelicobacter 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.