Differential activity of a lectin from Solieria filiformis against human pathogenic bacteria

A lectin isolated from the red alga Solieria filiformis was evaluated for its effect on the growth of 8 gram-negative and 3 gram-positive bacteria cultivated in liquid medium (three independent experiments/bacterium). The lectin (500 µg/mL) stimulated the growth of the gram-positive species Bacillus cereus and inhibited the growth of the gram-negative species Serratia marcescens, Salmonella typhi, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus sp, and Pseudomonas aeruginosa at 1000 µg/mL but the lectin (10-1000 µg/mL) had no effect on the growth of the gram-positive bacteria Staphylococcus aureus and B. subtilis, or on the gram-negative bacteria Escherichia coli and Salmonella typhimurium. The purified lectin significantly reduced the cell density of gram-negative bacteria, although no changes in growth phases (log, exponential and of decline) were observed. It is possible that the interaction of S. filiformis lectin with the cell surface receptors of gram-negative bacteria promotes alterations in the flow of nutrients, which would explain the bacteriostatic effect. Growth stimulation of the gram-positive bacterium B. cereus was more marked in the presence of the lectin at a concentration of 1000 µg/mL. The stimulation of the growth of B. cereus was not observed when the lectin was previously incubated with mannan (125 µg/mL), its hapten. Thus, we suggest the involvement of the binding site of the lectin in this effect. The present study reports the first data on the inhibition and stimulation of pathogenic bacterial cells by marine alga lectins.

Enucleated L929 mouse fibroblasts support invasion and multiplication of Shigella flexneri 5a

Invasive bacteria can induce their own uptake and specify their intracellular localization; hence it is commonly assumed that proximate modulation of host cell transcription is not required for infection. However, bacteria can also modulate, directly or indirectly, the transcription of many host cell genes, whose role in the infection may be difficult to determine by global gene expression. Is the host cell nucleus proximately required for intracellular infection and, if so, for which pathogens and at what stages of infection? Enucleated cells were previously infected with Toxoplasma gondii, Chlamydia psittaci, C. trachomatis, or Rickettsia prowazekii. We enucleated L929 mouse fibroblasts by centrifugation in the presence of cytochalasin B, and compared the infection with Shigella flexneri M90T 5a of nucleated and enucleated cells. Percent infection and bacterial loads were estimated with a gentamicin suppression assay in cultures fixed and stained at different times after infection. Enucleation reduced by about half the percent of infected cells, a finding that may reflect the reduced endocytic ability of L929 cytoplasts. However, average numbers of bacteria and frequency distributions of bacterial numbers per cell at different times were similar in enucleated and nucleated cells. Bacteria with actin-rich tails were detected in both cytoplasts and nucleated cells. Lastly, cytoplasts were similarly infected 2 and 24 h after enucleation, suggesting that short-lived mRNAs were not involved in the infection. Productive S. flexneri infection could thus take place in cells unable to modulate gene transcription, RNA processing, or nucleus-dependent signaling cascades.

Ureases display biological effects independent of enzymatic activity: Is there a connection to diseases caused by urease-producing bacteria?

Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

Isolation and intracellular localization of insulin-like proteins from leaves of Bauhinia variegata

Evidence based on immunological cross-reactivity and anti-diabetic properties has suggested the presence of insulin-like peptides in plants. The objective of the present study was to investigate the presence of insulin-like proteins in the leaves of Bauhinia variegata ("pata-de-vaca", "mororó"), a plant widely utilized in popular medicine as an anti-diabetic agent. We show that an insulin-like protein was present in the leaves of this plant. A chloroplast protein with a molecular mass similar to that of bovine insulin was extracted from 2-mm thick 15% SDS-PAGE gels and fractionated with a 2 x 24 cm Sephadex G-50 column. The activity of this insulin-like protein (0.48 mg/mL) on serum glucose levels of four-week-old Swiss albino (CF1) diabetic mice was similar to that of commercial swine insulin used as control. Further characterization of this molecule by reverse-phase hydrophobic HPLC chromatographic analysis as well as its antidiabetic activity on alloxan-induced mice showed that it has insulin-like properties. Immunolocalization of the insulin-like protein in the leaves of B. variegata was performed by transmission electron microscopy using a polyclonal anti-insulin human antibody. Localization in the leaf blades revealed that the insulin-like protein is present mainly in chloroplasts where it is also found associated with crystals which may be calcium oxalate. The presence of an insulin-like protein in chloroplasts may indicate its involvement in carbohydrate metabolism. This finding has strengthened our previous results and suggests that insulin-signaling pathways have been conserved through evolution.

NMDA receptor blockade alters the intracellular distribution of neuronal nitric oxide synthase in the superficial layers of the rat superior colliculus

Nitric oxide (NO) is a molecular messenger involved in several events of synaptic plasticity in the central nervous system. Ca2+ influx through the N-methyl-D-aspartate receptor (NMDAR) triggers the synthesis of NO by activating the enzyme neuronal nitric oxide synthase (nNOS) in postsynaptic densities. Therefore, NMDAR and nNOS are part of the intricate scenario of postsynaptic densities. In the present study, we hypothesized that the intracellular distribution of nNOS in the neurons of superior colliculus (SC) superficial layers is an NMDAR activity-dependent process. We used osmotic minipumps to promote chronic blockade of the receptors with the pharmacological agent MK-801 in the SC of 7 adult rats. The effective blockade of NMDAR was assessed by changes in the protein level of the immediate early gene NGFI-A, which is a well-known NMDAR activity-dependent expressing transcription factor. Upon chronic infusion of MK-801, a decrease of 47% in the number of cells expressing NGFI-A was observed in the SC of treated animals. Additionally, the filled dendritic extent by the histochemical product of nicotinamide adenine di-nucleotide phosphate diaphorase was reduced by 45% when compared to the contralateral SC of the same animals and by 64% when compared to the SC of control animals. We conclude that the proper intracellular localization of nNOS in the retinorecipient layers of SC depends on NMDAR activation. These results are consistent with the view that the participation of NO in the physiological and plastic events of the central nervous system might be closely related to an NMDAR activity-dependent function.

Treatment of hemorrhagic shock with hypertonic saline solution modulates the inflammatory response to live bacteria in lungs

Shock and resuscitation render patients more susceptible to acute lung injury due to an exacerbated immune response to subsequent inflammatory stimuli. To study the role of innate immunity in this situation, we investigated acute lung injury in an experimental model of ischemia-reperfusion (I-R) followed by an early challenge with live bacteria. Conscious rats (N = 8 in each group) were submitted to controlled hemorrhage and resuscitated with isotonic saline (SS, 0.9% NaCl) or hypertonic saline (HS, 7.5% NaCl) solution, followed by intratracheal or intraperitoneal inoculation of Escherichia coli. After infection, toll-like receptor (TLR) 2 and 4 mRNA expression was monitored by RT-PCR in infected tissues. Plasma levels of tumor necrosis factor α and interleukins 6 and 10 were determined by ELISA. All animals showed similar hemodynamic variables, with mean arterial pressure decreasing to nearly 40 mmHg after bleeding. HS or SS used as resuscitation fluid yielded equal hemodynamic results. Intratracheal E. coli inoculation per se induced a marked neutrophil infiltration in septa and inside the alveoli, while intraperitoneal inoculation-associated neutrophils and edema were restricted to the interseptal space. Previous I-R enhanced lung neutrophil infiltration upon bacterial challenge when SS was used as reperfusion fluid, whereas neutrophil influx was unchanged in HS-treated animals. No difference in TLR expression or cytokine secretion was detected between groups receiving HS or SS. We conclude that HS is effective in reducing the early inflammatory response to infection after I-R, and that this phenomenon is achieved by modulation of factors other than expression of innate immunity components.

Anti-parasitic action and elimination of intracellular Toxoplasma gondii in the presence of novel thiosemicarbazone and its 4-thiazolidinone derivatives

Toxoplasma, which infects all eukaryotic cells, is considered to be a good system for the study of drug action and of the behavior of infected host cells. In the present study, we asked if thiosemicarbazone derivatives can be effective against tachyzoites and which morphological and ultrastructural features of host cells and parasites are associated with the destruction of Toxoplasma. The compounds were tested in infected Vero cell culture using concentration screens (0.1 to 20 mM). The final concentration of 1 mM was chosen for biological assay. The following results were obtained: 1) These new derivatives decreased T. gondii infection with an in vitro parasite IC50% of 0.2-0.7 mM, without a significant effect on host cells and the more efficient compounds were 2, 3 (thiosemicarbazone derivatives) and 4 (thiazolidinone derivative); 2) The main feature observed during parasite elimination was continuous morphological disorganization of the tachyzoite secretory system, progressive organelle vesiculation, and then complete disruption; 3) Ultrastructural assays also revealed that progressive vesiculation in the cytoplasm of treated parasites did not occur in the host cell; 4) Vesiculation inside the parasite resulted in death, but this feature occurred asynchronously in different intracellular tachyzoites; 5) The death and elimination of T. gondii was associated with features such as apoptosis-like stage, acidification and digestion of parasites into parasitophorous vacuoles. Our results suggest that these new chemical compounds are promising for the elimination of intracellular parasites by mainly affecting tachyzoite development at 1 mM concentration for 24 h of treatment.

Molecular battles between plant and pathogenic bacteria in the phyllosphere

The phyllosphere, i.e., the aerial parts of the plant, provides one of the most important niches for microbial colonization. This niche supports the survival and, often, proliferation of microbes such as fungi and bacteria with diverse lifestyles including epiphytes, saprophytes, and pathogens. Although most microbes may complete the life cycle on the leaf surface, pathogens must enter the leaf and multiply aggressively in the leaf interior. Natural surface openings, such as stomata, are important entry sites for bacteria. Stomata are known for their vital role in water transpiration and gas exchange between the plant and the environment that is essential for plant growth. Recent studies have shown that stomata can also play an active role in limiting bacterial invasion of both human and plant pathogenic bacteria as part of the plant innate immune system. As counter-defense, plant pathogens such as Pseudomonas syringae pv tomato (Pst) DC3000 use the virulence factor coronatine to suppress stomate-based defense. A novel and crucial early battleground in host-pathogen interaction in the phyllosphere has been discovered with broad implications in the study of bacterial pathogenesis, host immunity, and molecular ecology of bacterial diseases.

TRP channels, omega-3 fatty acids, and oxidative stress in neurodegeneration: from the cell membrane to intracellular cross-links

The transient receptor potential channels family (TRP channels) is a relatively new group of cation channels that modulate a large range of physiological mechanisms. In the nervous system, the functions of TRP channels have been associated with thermosensation, pain transduction, neurotransmitter release, and redox signaling, among others. However, they have also been extensively correlated with the pathogenesis of several innate and acquired diseases. On the other hand, the omega-3 polyunsaturated fatty acids (n-3 fatty acids) have also been associated with several processes that seem to counterbalance or to contribute to the function of several TRPs. In this short review, we discuss some of the remarkable new findings in this field. We also review the possible roles played by n-3 fatty acids in cell signaling that can both control or be controlled by TRP channels in neurodegenerative processes, as well as both the direct and indirect actions of n-3 fatty acids on TRP channels.

Diversity of 16S rRNA genes from bacteria of sugarcane rhizosphere soil

Sugarcane is an important agricultural product of Brazil, with a total production of more than 500 million tons. Knowledge of the bacterial community associated with agricultural crops and the soil status is a decisive step towards understanding how microorganisms influence crop productivity. However, most studies aim to isolate endophytic or rhizosphere bacteria associated with the plant by culture-dependent approaches. Culture-independent approaches allow a more comprehensive view of entire bacterial communities in the environment. In the present study, we have used this approach to assess the bacterial community in the rhizosphere soil of sugarcane at different times and under different nitrogen fertilization conditions. At the high taxonomic level, few differences between samples were observed, with the phylum Proteobacteria (29.6%) predominating, followed by Acidobacteria (23.4%), Bacteroidetes (12.1%), Firmicutes (10.2%), and Actinobacteria (5.6%). The exception was the Verrucomicrobia phylum whose prevalence in N-fertilized soils was approximately 0.7% and increased to 5.2% in the non-fertilized soil, suggesting that this group may be an indicator of nitrogen availability in soils. However, at low taxonomic levels a higher diversity was found associated with plants receiving nitrogen fertilizer. Bacillus was the most predominant genus, accounting for 19.7% of all genera observed. Classically reported nitrogen-fixing and/or plant growth-promoting bacterial genera, such as Azospirillum, Rhizobium, Mesorhizobium, Bradyrhizobium, and Burkholderia were also found although at a lower prevalence.

Resveratrol inhibits the intracellular calcium increase and angiotensin/endothelin system activation induced by soluble uric acid in mesangial cells

Resveratrol (Resv) is natural polyphenol found in grapes. This study evaluated the protective effect of Resv against the effects of uric acid (UA) in immortalized human mesangial cells (ihMCs). ihMCs were preincubated with Resv (12.5 µM) for 1 h and treated with UA (10 mg/dL) for 6 or 12 h. The intracellular calcium concentration [Ca2+]i was quantified by fluorescence using flow cytometry. Angiotensinogen (AGT) and pre-pro endothelin-1 (ppET-1) mRNA were assayed by quantitative real-time RT-PCR. Angiotensin II (AII) and endothelin-1 (ET-1) were assayed by ELISA. UA significantly increased [Ca2+]i. Pre-incubation with Resv significantly reduced the change in [Ca2+]i induced by UA. Incubation with UA for 6 or 12 h also increased AGT mRNA expression and AII protein synthesis. Resv blunted these increases in AGT mRNA expression and AII protein. Incubation with UA in the ihMCs increased ppET-1 expression and ET-1 protein synthesis at 6 and 12 h. When ihMCs were pre-incubated with Resv, UA had a significantly diminished effect on ppET-1 mRNA expression and ET-1 protein synthesis at 6 and 12 h, respectively. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. The renin-angiotensin system may contribute to the pathogenesis of renal function and chronic kidney disease. Resv can minimize the impact of UA on AII, ET-1 and the increase of [Ca2+]i in mesangial cells, suggesting that, at least in part, Resv can prevent the effects of soluble UA in mesangial cells.

Evaluation of thermotolerant capacity of lactic acid bacteria isolated from commercial sausages and the effects of their addition on the quality of cooked sausages

The thermotolerant capacity of several lactic acid bacteria strains isolated from cooked commercial sausages was determined. Four strains were positively identified as Lactobacillus plantarum, Lactobacillus curvatus, Pediococcus pentosaceus and Pediococcus acidilacti, after surviving thermal treatment (70 °C during 60 minutes). Thermotolerant strains were inoculated in sausage batters before cooking in order to determine their effect on color, texture, acceptance and inhibition of Enterobacteria during 12 days at 8 °C. No significant effect of the inoculated strains was detected on color parameters. Textural profile parameters, cohesiveness and resilience, were not affected by the inoculation of thermotolerant lactic acid bacteria, but L. curvatus sausages resulted softer than the rest of the treatments. Samples inoculated with L. curvatus also obtained the lowest scores for the sensory attributes evaluated, with the remaining treatments causing no unfavorable effects on sausage acceptance. There was a reduction in enterobacterial counts after 12 days of cold storage in inoculated samples. The performance of inoculated lactic acid bacteria strains can be explained in a similar way as that of starter cultures in dry-fermented sausages, where the growth in nests impairs other pathogenic microorganisms present in the rest of the sausage, since environmental conditions and the early inoculation of these thermotolerant strains favor them to become the dominant flora.

Study of bacteria Gluconobacter sp.: isolation, purification, phenotypic and molecular identification

The importance of the study of acetic bacteria, on species of the Gluconobacter genus is based on its industrial application, as these possess the capacity of bioconversion of sorbitol to sorbose, enabling the process of vitamin C production. The study involved samples collected in industries of soft drinks, flowers, fruits and honey, followed by purification, phenotypic identification, molecular identification with the use of primer defined from Nucleotide Sequence Database consultation. Strains preserved were identified as members of the Acetobacteraceae family, Gluconobacter genus. 110 strains had been isolated of substrate: Pyrostegia venusta (ker-gawler), honey, Vitis vinifera (grape), Pyrus communis (pear), Malus sp. (apple) and in two samples of soft drinks. Of this total 57 strains had been recovered in manitol medium (manitol, yeast extract, peptone), 12 in YMG medium (glucose, manitol, yeast extract, ethanol, acetic acid), 41 in enrichment medium (De Ley and Swings) and later in the GYC medium (glucose, yeast extract and calcium carbonate). 68 strains were identified as Gram negative bacilli rods. Of these, 31 were characterized biochemically as belonging to the Acetobacteriaceae family as they were catalase positive, oxidase negative and producers of acid from glucose. The characterization of these strains was complemented with the biochemistry tests: gelatin liquefaction, nitrate reduction, indole and H2S production, oxidation of ethanol to acetic acid and molecular tests for genus identification. Only eight strains were characterized as pertaining to the Gluconobacter genus. The strains are maintained in collection cultures at the Microbiology Laboratory of the Biology Department at the São Paulo State University (UNESP) in Assis, stored in malt extract at -196 ºC.

Inhibitory effect of the essential oil from Cinnamomum zeylanicum Blume leaves on some food-related bacteria

Cinnamomum zeylanicum Blume, Lauraceae, has long been known for having many biological properties. This study aimed to identify the constituents of the essential oil from C. zeylanicum leaves using GC-MS and to assess its inhibitory effect on Salmonella enterica, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa based on MIC and MBC determination and kill-time study. Eugenol (73.27%) was the most prevalent compound in the essential oil followed by trans-β-cariophyllene (5.38%), linalool (3.31%), and alcohol cinamic acetate (2.53%). The results showed an interesting antibacterial activity of the oil with MIC ranging from 1.25 to 10 µL.mL-1. MBC values were in the range of 20 - 80 µL.mL-1. A concentration of 10 and 40 µL.mL-1 of the essential oil caused a fast and steady decrease in viable cell count (2 to 5 log cycles) of all assayed strains along 24 hours. A concentration of 40 µL.mL-1 of the oil provided a total elimination of the initial inocula of S. aureus after 2 hours. These results show the possibility of regarding the essential oil from C. zeylanicum leaves as alternative sources of antimicrobial compounds to be applied in food conservation systems.

Culture alternative medium for the growth of extreme halophilic bacteria in fish products

Halotolerant or halophilic (Archaeabacteria) microorganisms can be found in salted and ripening fish products that are not affected by salt. They can be moderate or extremely halophilic bacteria. The extremely halophilic bacteria require between 15-30% of NaCl for growth. The extremely halophilic archaeobacteria may be selectively isolated in different media. The aim of this work was to determine the effectiveness of the Salt-Agar-Milk medium, a medium modified in our laboratory through the addition of MgSO4 and KCl - named SAMm, and its effect on the bacterial growth by means of comparison with other media, with and without milk, determining time of incubation and counting. Two samples of salted fish from local fish salting factories and two laboratory strains were used. The factory samples were matured anchovy and anchovy fillets in oil, and the laboratory strains were: Haloarcula spp. (proteolytic) and Halococcus spp. (non-proteolytic). The following media were alternatively used for the isolation of extremely halophilic bacteria: IRAM; Formulation of Gibbons and collaborators, Cod Milk agar, and SAMm. IRAM and Gibbons were also used enriched with milk. In the SAMm medium, there were obtained count values similar or higher than the ones of the traditional media; besides the simplicity of its elaboration, the possibility to obtain positive results two or three days earlier also added to its benefit. Consequently, it can be considered an alternative to the media traditionally used for the studied halophilic bacteria.