Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.Fondo Especial de la Educación Superior/[0500-13]/FEES/Costa RicaFondo Especial de la Educación Superior/[0504-13]/FEES/Costa RicaFondo Especial de la Educación Superior/[0505-13]/FEES/Costa RicaFondo Especial de la Educación Superior/[0248-13]/FEES/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Influence of alkali treatment on agar from Gracilariopsis longissima and Gracilaria vermiculophylla from the Gulf of California, Mexico

The effect of alkali treatment on agar yield, sulfate content, and 3,6 anhydrogalactose content from two agarophyte species (Gracilariopsis longissima and Gracilaria vermiculophylla) was analyzed. In addition, gel strength was measured in each agar extract as well as gelling and melting temperatures. Agar was extracted after pretreatment with different concentrations of NaOH (3%, 5%, 7%, and 9%). Pretreatment with alkali significantly reduced agar yield from G. longissima from 13.2% in nontreated agar (native agar) to 5.4% in 5% NaOH, whereas in G. vermiculophylla it diminished from 25.2% to 9.6% in 9% NaOH treatment. Statistically significant differences were observed for native and all alkali treatments in G. longissima, whereas in G. vermiculophylla significant differences were only found among native and high alkali treatments (7% and 9% NaOH, P < 0.05). Sulfate content from G. longissima diminished gradually with increasing alkali concentration, from high (3.9%) in native agar to low (3.1%) in 7% alkali treatment, and was negatively correlated with gel strength (r = –0.92). Sulfate content in G. vermiculophylla native agar was twice as high as that found in G. longissima (7.8%) and did not show statistically significant differences with low alkali treatments, but it did with high NaOH treatments. Higher contents of 3,6 anhydrogalactose were found with 9% NaOH treatment in G. longissima (40.9%) and G. vermiculophylla (43%), and they were significantly correlated with gel strength (r = 0.89 and r = 0.91, respectively).

Structural, physical, and chemical modifications induced by microwave heating on native agar-like galactans

Native agars from Gracilaria vermiculophylla produced in sustainable aquaculture systems (IMTA) were extracted under conventional (TWE) and microwave (MAE) heating. The optimal extracts from both processes were compared in terms of their properties. The agars’ structure was further investigated through Fourier transform infrared and NMR spectroscopy. Both samples showed a regular structure with an identical backbone, β-D-galactose (G) and 3,6-anhydro-α-L-galactose (LA) units; a considerable degree of methylation was found at C6 of the G units and, to a lesser extent, at C2 of the LA residues. The methylation degree in the G units was lower for MAEopt agar; the sulfate content was also reduced. MAE led to higher agar recoveries with drastic extraction time and solvent volume reductions. Two times lower values of [η] and Mv obtained for the MAEopt sample indicate substantial depolymerization of the polysaccharide backbone; this was reflected in its gelling properties; yet it was clearly appropriate for commercial application in soft-texture food products

Production and properties of agar from the invasive marine alga, Gracilia Vermiculophylla (Gracilariales, Rhodophyta)

The utilization potential, in terms of agar production, of the invasive alga, Gracilaria vermiculophylla, collected at Ria de Aveiro, northwestern Portugal was investigated. The agar yield ranged from 15% to 33%, with pre-extraction treatment with alkali generally increasing the yield. The gel quality (gel strength and apparent Young’s modulus) was best (>600 g cm−2 and >1,000 kPa, respectively) when alkali treatment with 6% NaOH for 3.5 h was performed. At these pretreatment conditions, the effect of extraction time was also investigated and highest yield and best gel quality were obtained with a 2 h extraction time. By employing these extraction conditions, G. vermiculophylla can be a source of industrial food-grade agar. The structure of agar from G. vermiculophylla was determined through chemical techniques and FTIR and NMR spectrometry. It is mainly composed of alternating 3-linked D-galactose and 4-linked 3,6-anhydro-L-galactose, with methyl substitution occurring at 16–19 mol% of C-6 in 3-linked units and 2–3 mol% of C-2 in 4-linked units. A minor sulfation on C-4 of 3-linked units was also detected; while precursor units (6-sulfated 4-linked galactosyl moieties) were found in the native extract.We thank R. Pereira for algal material collection, L. Martins for FTIR analysis, and two anonymous reviewers for valuable comments on the manuscript. RDV was supported by the Portuguese Foundation for Science and Technology, through a post-doctoral grant (SFRH/BPD/34670/2007)