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SummaryThe constant arms race between bacteria and their phages has resulted in a large diversity of bacterial defence systems1,2, with many bacteria carrying several systems3,4. In response, phages often carry counter-defence genes5–9. If and how bacterial defence mechanisms interact to protect against phages with counter-defence genes remains unclear. Here, we report the existence of a novel defence system, coined MADS (Methylation Associated Defence System), which is located in a strongly conserved genomic defence hotspot inPseudomonas aeruginosaand distributed across Gram-positive and Gram-negative bacteria. We find that the natural co-existence of MADS and a Type IE CRISPR-Cas adaptive immune system in the genome ofP. aeruginosaSMC4386 provides synergistic levels of protection against phage DMS3, which carries an anti-CRISPR (acr) gene. Previous work has demonstrated that Acr-phages need to cooperate to overcome CRISPR immunity, with a first sacrificial phage causing host immunosuppression to enable successful secondary phage infections10,11. Modelling and experiments show that the co-existence of MADS and CRISPR-Cas provides strong and durable protection against Acr-phages by disrupting their cooperation and limiting the spread of mutants that overcome MADS. These data reveal that combining bacterial defences can robustly neutralise phage with counter-defence genes, even if each defence on its own can be readily by-passed, which is key to understanding how selection acts on defence combinations and their coevolutionary consequences.

Type VI Secretion Systems (T6SSs) are widespread in bacteria and can dictate the development and organisation of polymicrobial ecosystems by mediating contact dependent killing. In Neisseria species, including Neisseria cinerea a commensal of the human respiratory tract, interbacterial contacts are mediated by Type four pili (Tfp) which promote formation of aggregates and govern the spatial dynamics of growing Neisseria microcolonies. Here, we show that N. cinerea expresses a plasmid-encoded T6SS that is active and can limit growth of related pathogens. We explored the impact of Tfp on N. cinerea T6SS-dependent killing within a colony and show that pilus expression by a prey strain enhances susceptibility to T6SS compared to a non-piliated prey, by preventing segregation from a T6SS-wielding attacker. Our findings have important implications for understanding how spatial constraints during contact-dependent antagonism can shape the evolution of microbial communities.

Type VI Secretion Systems (T6SS) are widespread in bacteria and can dictate the development and organisation of polymicrobial ecosystems by mediating contact dependent killing. In Neisseria species, including Neisseria cinerea a commensal of the human respiratory tract, interbacterial contacts are mediated by Type four pili (Tfp) which promote formation of aggregates and govern the spatial dynamics of growing Neisseria microcolonies. Here we show that N. cinerea expresses a plasmid-encoded T6SS that is active and can limit growth of related pathogens. We explored the impact of Tfp expression on N. cinerea T6SS-dependent killing and show that expression of Tfp by prey strains enhances their susceptibility to T6SS, by keeping them in close proximity of T6SS-wielding attacker strains. Our findings have important implications for understanding how spatial constraints during contact-dependent antagonism can shape the evolution of microbial communities.

Type VI Secretion Systems (T6SSs) are widespread in bacteria and can dictate the development and organisation of polymicrobial ecosystems by mediating contact dependent killing. In Neisseria species, including Neisseria cinerea a commensal of the human respiratory tract, interbacterial contacts are mediated by Type four pili (Tfp) which promote formation of aggregates and govern the spatial dynamics of growing Neisseria microcolonies. Here, we show that N. cinerea expresses a plasmid-encoded T6SS that is active and can limit growth of related pathogens. We explored the impact of Tfp on N. cinerea T6SS-dependent killing within a colony and show that pilus expression by a prey strain enhances susceptibility to T6SS compared to a non-piliated prey, by preventing segregation from a T6SS-wielding attacker. Our findings have important implications for understanding how spatial constraints during contact-dependent antagonism can shape the evolution of microbial communities.

It is increasingly being recognised that the interplay between commensal and pathogenic bacteria can dictate the outcome of infection. Consequently, there is a need to understand how commensals interact with their human host and influence pathogen behaviour at epithelial surfaces. Neisseria meningitidis, a leading cause of sepsis and meningitis, exclusively colonises the human nasopharynx and shares this niche with several other Neisseria species, including the commensal Neisseria cinerea. Here, we demonstrate that during adhesion to human epithelial cells N. cinerea co-localises with molecules that are also recruited by the meningococcus, and show that, similar to N. meningitidis, N. cinerea forms dynamic microcolonies on the cell surface in a Type four pilus (Tfp) dependent manner. Finally, we demonstrate that N. cinerea colocalises with N. meningitidis on the epithelial cell surface, limits the size and motility of meningococcal microcolonies, and impairs the effective colonisation of epithelial cells by the pathogen. Our data establish that commensal Neisseria can mimic and affect the behaviour of a pathogen on epithelial cell surfaces., Author summary Commensal and pathogenic bacteria can establish long term relationships with their hosts. Despite this, very little is known about the processes of attachment, replication and organisation of commensal bacteria at epithelial surfaces. In this work, we have examined how Neisseria cinerea, a typically commensal species that colonises the human nasopharynx similar to the closely-related pathogen Neisseria meningitidis, engages with human epithelial cells. We show that N. cinerea on human epithelial cells mimics some of the behaviour of the meningococcus, but have identified subtle differences that distinguish the two species. Furthermore, we show that the presence of N. cinerea affects the interaction of N. meningitidis with epithelial cells, providing evidence that the interaction between two closely related species can affect pathogen colonisation of the epithelial surface.

Orientador: Sueli Irene Rodrigues Costa Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Matemática Estatística e Computação Científica Resumo: Nesta dissertação abordamos reticulados e suas conexões com códigos corretores de erros. Reticulados são conjuntos discretos de pontos em um espaço euclidiano n-dimensional, que são descritos como todas as combinações inteiras de vetores linearmente independentes. Eles têm sido estudados em matemática devido a sua simetria e propriedades especiais, na área de comunicações, dadas suas aplicações em codificação para transmissão de sinais em diferentes canais, e também em criptografia. No trabalho aqui apresentado estudamos reticulados construídos a partir de uma classe bem conhecida de códigos, os códigos cíclicos, e também de códigos binários duplamente cíclicos que foram introduzidos recentemente. Analisamos particularmente a densidade de empacotamento destes reticulados Abstract: In this dissertation we address lattices and their connections with error correcting codes. Lattices are discrete sets of points in an n-dimensional Euclidean space, which are described as all integer combinations of linearly independent vectors. They have been studied in mathematics due to their symmetry and special properties, in the area of communications, due to their applications in coding for signal transmission trought different channels and also in cryptografy. In the work presented here, we study lattices built from a well-known class of codes, the cyclic codes, and also from double binary cyclic codes which were recently introduced. In particular we analyze the packing density of these lattices Mestrado Matemática Mestre em Matemática CNPQ 143171/2018-6

This month’s Under the Lens discusses electron cryotomography (ECT) as a powerful tool for investigating host–pathogen interactions. This month’s Under the Lens discusses electron cryotomography (ECT) as a powerful tool for investigating host–pathogen interactions.

Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signalling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signalling molecule is sphingosine-1-phosphate (S1P) whose breakdown is catalysed by sphingosine-1-phosphate lyase (S1PL), a pyridoxal 5 ′-phosphate (PLP) dependent enzyme that catalyses the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine (PE). Here we show the pathogenic bacterium Burkholderia pseudomallei K96243 encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established mass spectrometry-based methodology we show that recombinant S1PL2021 is catalytically active. Using recombinant human fatty aldehyde dehydrogenase (FALDH) we developed a spectrophotometric, enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the x-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in the orchestration of immune responses. S1P levels within the mammalian host are tightly regulated, in part through the activity of S1P lyase (S1PL) which catalyses its irreversible degradation. Herein, we describe the identification and characterization of secreted S1PL orthologues encoded by the facultative intracellular bacteria Burkholderia pseudomallei and Burkholderia thailandensis. These bacterial orthologues exhibited S1PL enzymatic activity, functionally complemented an S1PL-deficient yeast strain and conferred resistance to the antimicrobial sphingolipid D-erythro-sphingosine. We report that secretion of these bacterial S1PLs is pH-dependent, and is observed during intracellular infection. S1PL-deficient mutants displayed impaired intracellular replication in murine macrophages (associated with an inability to evade the maturing phagosome) and were significantly attenuated in murine and larval infection models. Furthermore, treatment of Burkholderia-infected macrophages with either S1P or a selective agonist of S1P receptor 1 enhanced bacterial colocalisation with LAMP-1 and reduced their intracellular survival. In summary, our studies confirm bacterial-encoded S1PL as a critical virulence determinant of B. pseudomallei and B. thailandensis, further highlighting the pivotal role of S1P in host-pathogen interactions. In addition, our data suggest that S1P pathway modulators have potential for the treatment of intracellular infection.

Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signaling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signaling molecule is sphingosine-1-phosphate (S1P), whose breakdown is catalyzed by S1P lyase (S1PL), a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine. Here, we show that the pathogenic bacterium, Burkholderia pseudomallei K96243, encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established MS-based methodology, we show that recombinant S1PL2021 is catalytically active. We also used recombinant human fatty aldehyde dehydrogenase to develop a spectrophotometric enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the X-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing that the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.

Objetivo. Evaluar el efecto antinociceptivo agudo y sobre la neuroconducta de la amitriptilina en dosis escalonadas. Materiales y métodos. Se utilizaron 82 ratones de aproximadamente 28 g, distribuidos en 10 grupos; se les suministró vía oral: agua destilada (0,1 mL/10 g), tramadol (20 mg/Kg), cafeína (32 mg/Kg), diazepam (32 mg/Kg)y amitriptilina en dosis escalonada (10-50 mg/Kg). Se emplearon las pruebas de la "placa caliente" y de Irwin. Se aplicaron las pruebas estadísticas de ANOVA, Tukey, coeficiente de correlación de Pearson y la exacta de Fisher. Resultados. La amitriptilina incrementó el umbral del dolor según el incremento de la dosis administrada, de 30 a 50 mg/Kg (p < 0,05; IC 95%). En las mismas dosis, se observó la presencia de sedación en 46,9%, 56,2%, y 81,2%, respectivamente. Conclusiones. Se demostró la acción antinociceptiva aguda de la amitriptilina y su relación dosis-respuesta en el rango de 30-50 mg/Kg, asimismo, se describieron manifestaciones neuroconductuales como: sedación, piloerección, incoordinación motora, salto, respiración anormal y marcha anormal. Objective. To evaluate the antinociceptive effect in acute pain and neurobehavioral of amitriptyline in dose response. Materials and methods. It was used 82 mice of approximately 28 g, distributed in 10 groups. It was given by orally: distilled water (0.1 ml/10 g), tramadol (20 mg/Kg), caffeine (32 mg/Kg), diazepam (32 mg/Kg), and amitriptyline, in doses staggered (10 to 50 mg/Kg). These were used the hot plate test and the Irwin test. The statistics validation was made trougth the next test: one way ANOVA, Tukey, Pearson correlation coefficient and the Fisher exact test. Results. Amitriptyline increased the pain threshold by increasing the administered dose of 30 to 50 mg / kg (p

The production curve of the main Mexican offshore fields has been decreasing rapidly year-on-year, representing a challenge to the eighth-largest oil producer country and an opportunity to implement new techniques for workover (WO) operations to increase production recovery.Within the most common WO activities developed in offshore Mexico is acid stimulation of carbonates, which was developed as pumping a large volume of fluids (solvents, HCl 15%, etc.) with minimum control for zonal coverage. Conventional coiled tubing (CT) jobs have improved this technique with the use of impact jetting tools to remove damage at the tubular and perforation holes and placing the treatment in the desired area. The estimation of fluid injection per zone has been evaluated using radioactive traces. However, the estimates of where the fluids go lack accuracy, and the radioactivity poses additional risks related to hazardous material handling.In 2009, a new interpretation technique to evaluate the treatment zonal coverage appeared that used CT with fiber optics. This distributed temperature sensing (DTS) enables the equipment to record temperature readings at several predetermined depths over time creating a survey that identifies, qualitatively, the admittance of the treated pay zones based on the formation’s cold-down/heat-up effects. Unfortunately, this technique had some limitations as the data recorded needed to be converted, interpreted, and then translated into common oilfield language in the form of graphics and data tables. The time to get a final result from the readings to the customer was so long that the window to modify the current treatment to improve the well response was, on most occasions, already closed.DTS inversion brings a "real-time DTS matrix stimulation evaluation product" by using an inversion algorithm coupled with a fluid placement model to accurately quantify the amount of treatment placed across the treated zones of the well. A detailed case history from an offshore Mexico well describes the first worldwide real-time DTS inversion job and illustrates the workflow and advantages of this technique. The results from the technique showed a direct match with a previous evaluation of the formation through production logging conveyed on Wireline tools.