Type IV secretion system is not involved in infection process in citrus

The type IV secretion system (T4SS) is used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Xanthomonas citri subsp. citri contains two copies of the T4SS, one in the chromosome and the other is plasmid-encoded. To understand the conditions that induce expression of the T4SS in Xcc, we analyzed, in vitro and in planta, the expression of 18 ORFs from the T4SS and 7 hypothetical flanking genes by RT-qPCR. As a positive control, we also evaluated the expression of 29 ORFs from the type III secretion system (T3SS), since these genes are known to be expressed during plant infection condition, but not necessarily in standard culture medium. From the 29 T3SS genes analyzed by qPCR, only hrpA was downregulated at 72 h after inoculation. All genes associated with the T4SS were downregulated on Citrus leaves 72 h after inoculation. Our results showed that unlike the T3SS, the T4SS is not induced during the infection process.

Neutrophil extracellular traps identification in tegumentary lesions of patients with paracoccidioidomycosis and different patterns of NETs generation in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Jato de plasma frio em pressão atmosférica para tratamento de materiais e esterilização

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo comparativo de cápsulas contendo amoxicilina obtidas de algumas farmácias magistrais

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Indução diferencial das vias MyD88 e TRIF-dependentes em leucócitos totais de equinos estimulados com LPS de E. coli

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Associação de antibióticos e terapia fotodinâmica antimicrobiana para o controle de Acinetobacter baumannii

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo químico e avaliação da atividade antimicrobiana de fungos endofíticos associados à Paepalanthus chiquitensis (eriocaulaceae) e efeitos de moduladores químicos epigenéticos no cultivo de Pochonia chlamydosporia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of Heme Oxygenase-1 in Polymyxin B-Induced Nephrotoxicity in Rats

Polymyxin B (PMB) is a cationic polypeptide antibiotic with activity against multidrug-resistant Gram-negative bacteria. PMB-induced nephrotoxicity consists of direct toxicity to the renal tubules and the release of reactive oxygen species (ROS) with oxidative damage. This study evaluated the nephroprotective effect of heme oxygenase-1 (HO-1) against PMB-induced nephrotoxicity in rats. Adult male Wistar rats, weighing 286 +/- 12 g, were treated intraperitoneally once a day for 5 days with saline, hemin (HO-1 inducer; 10 mg/kg), zinc protoporphyrin (ZnPP) (HO-1 inhibitor; 50 mu mol/kg, administered before PMB on day 5), PMB (4 mg/kg), PMB plus hemin, and PMB plus ZnPP. Renal function (creatinine clearance, Jaffe method), urinary peroxides (ferrous oxidation of xylenol orange version 2 [FOX-2]), urinary thiobarbituric acid-reactive substances (TBARS), renal tissue thiols, catalase activity, and renal tissue histology were analyzed. The results showed that PMB reduced creatinine clearance (P < 0.05), with an increase in urinary peroxides and TBARS. The PMB toxicity caused a reduction in catalase activity and thiols (P < 0.05). Hemin attenuated PMB nephrotoxicity by increasing the catalase antioxidant activity (P < 0.05). The combination of PMB and ZnPP incremented the fractional interstitial area of renal tissue (P < 0.05), and acute tubular necrosis in the cortex area was also observed. This is the first study demonstrating the protective effect of HO-1 against PMB-induced nephrotoxicity.

Sardinian goat's milk as source of bacteriocinogenic potential protective cultures

Goat breeding in Sardinia constitutes an important source of income for farming and shepherding activities. In this study 170 LAB strains were isolated from Sardinian goat's milk and tested for bacteriocins production against several food-borne pathogenic microorganisms. Four isolates (SD1, SD2, SD3 and SD4) were selected for their effective inhibition on Listeria monocytogenes. The strains were classified as members of Enterococcus genus, according to their biochemical and physiological characteristics, and then genetically identified as Enterococcus faecium. In MRS broth at 37 degrees C, bacteriocins SD1 and SD2 were produced at much higher levels (51200 AU/ml) compared to bacteriocin SD3 (3200 AU/ml) and bacteriocin SD4 (800 AU/ml). Their peptides were inactivated by proteolytic enzymes, but not when treated with alpha-amylase, catalase and lipase. The four bacteriocins remained stable at pH from 2.0 to 12.0, after exposure to 100 degrees C for 120 min and were not affected by the presence of surfactants and salts (N-Laourylsarcosine, NaCl, SDS, Triton X-100, Tween 20, Tween 80 and urea). Their molecular size was determined to be approximately 5 kDa by tricine-SDS-PAGE. Since the strains exhibited a strong antimicrobial activity against 21 L monocytogenes strains and 6 Salmonella spp. isolates, they should be considered as potential bio-preservatives cultures for fermented food productions. Moreover, due to their technological features, the four strains could be taken in account for using as adjunct NSLAB (non-starter lactic acid bacteria) rather than as starter culture. (C) 2011 Elsevier Ltd. All rights reserved.

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coil

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa. (C) 2012 Elsevier Inc. All rights reserved.

Hypoactivity of the central dopaminergic system and autistic-like behavior induced by a single early prenatal exposure to lipopolysaccharide

The aim of the present study was to evaluate the behavioral patterns associated with autism and the prevalence of these behaviors in males and females, to verify whether our model of lipopolysaccharide (LPS) administration represents an experimental model of autism. For this, we prenatally exposed Wistar rats to LPS (100 mu g/kg, intraperitoneally, on gestational day 9.5), which mimics infection by gram-negative bacteria. Furthermore, because the exact mechanisms by which autism develops are still unknown, we investigated the neurological mechanisms that might underlie the behavioral alterations that were observed. Because we previously had demonstrated that prenatal LPS decreases striatal dopamine (DA) and metabolite levels, the striatal dopaminergic system (tyrosine hydroxylase [TH] and DA receptors D1a and D2) and glial cells (astrocytes and microglia) were analyzed by using immunohistochemistry, immunoblotting, and real-time PCR. Our results show that prenatal LPS exposure impaired communication (ultrasonic vocalizations) in male pups and learning and memory (T-maze spontaneous alternation) in male adults, as well as inducing repetitive/restricted behavior, but did not change social interactions in either infancy (play behavior) or adulthood in females. Moreover, although the expression of DA receptors was unchanged, the experimental animals exhibited reduced striatal TH levels, indicating that reduced DA synthesis impaired the striatal dopaminergic system. The expression of glial cell markers was not increased, which suggests that prenatal LPS did not induce permanent neuroinflammation in the striatum. Together with our previous finding of social impairments in males, the present findings demonstrate that prenatal LPS induced autism-like effects and also a hypoactivation of the dopaminergic system. (c) 2012 Wiley Periodicals, Inc.

Comparative Genomics of Early-Diverging Brucella Strains Reveals a Novel Lipopolysaccharide Biosynthesis Pathway

Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1(T) and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1(T) and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1(T) and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and Mantisera against O-side chain sugars composed of N-formyl-perosamine. While BO1(T) maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. IMPORTANCE This report examines differences between genomes from four new Brucella strains and those from the classic Brucella spp. Our results show that the four new strains are outliers with respect to the previously known Brucella strains and yet are part of the genus, forming two new clades. The analysis revealed important information about the evolution and survival mechanisms of Brucella species, helping reshape our knowledge of this important zoonotic pathogen. One discovery of special importance is that one of the strains, BO2, produces an O-antigen distinct from any that has been seen in any other Brucella isolates to date.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coil BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K-D) of 292 +/- 24 nM and 157 +/- 35 nM, respectively. Moreover, the Lsa30 is a plasminogen (PLC) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. (C) 2012 Elsevier Ltd. All rights reserved.

Lipopolysaccharide-Induced Sickness Behaviour Evaluated in Different Models of Anxiety and Innate Fear in Rats

The fact that there is a complex and bidirectional communication between the immune and nervous systems has been well demonstrated. Lipopolysaccharide (LPS), a component of gram-negative bacteria, is widely used to systematically stimulate the immune system and generate profound physiological and behavioural changes, also known as sickness behaviour (e.g. anhedonia, lethargy, loss of appetite, anxiety, sleepiness). Different ethological tools have been used to analyse the behavioural modifications induced by LPS; however, many researchers analysed only individual tests, a single LPS dose or a unique ethological parameter, thus leading to disagreements regarding the data. In the present study, we investigated the effects of different doses of LPS (10, 50, 200 and 500 mu g/kg, i.p.) in young male Wistar rats (weighing 180200 g; 89 weeks old) on the ethological and spatiotemporal parameters of the elevated plus maze, light-dark box, elevated T maze, open-field tests and emission of ultrasound vocalizations. There was a dose-dependent increase in anxiety-like behaviours caused by LPS, forming an inverted U curve peaked at LPS 200 mu g/kg dose. However, these anxiety-like behaviours were detected only by complementary ethological analysis (stretching, grooming, immobility responses and alarm calls), and these reactions seem to be a very sensitive tool in assessing the first signs of sickness behaviour. In summary, the present work clearly showed that there are resting and alertness reactions induced by opposite neuroimmune mechanisms (neuroimmune bias) that could lead to anxiety behaviours, suggesting that misunderstanding data could occur when only few ethological variables or single doses of LPS are analysed. Finally, it is hypothesized that this bias is an evolutionary tool that increases animals security while the body recovers from a systemic infection.

A crucial role for IL-6 in the CNS of rats during fever induced by the injection of live E. coli

Interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, and IL-6 have been established as important mediators of fever induced by lipopolysaccharide (LPS) from Gram-negative bacteria. Whether these pro-inflammatory cytokines are also important in mediating fever induced by live bacteria remains less certain. We therefore investigated the following: (1) the synthesis of TNF-alpha, IL-1 beta, and IL-6 during E. coli-induced fever and (2) the effect of blocking the action of cytokines within the brain on E. coli-induced fever. Body or tail skin temperature (bT or Tsk, respectively) was measured by biotelemetry or telethermometry, every 30 min, during 6 or 24 h. Depending on the number of colony-forming units (CFU) injected i.p., administration of E. coli induced a long-lasting increase in bT of male Wistar rats. The duration of fever did not correlate with the number of CFU found in peritoneal cavity or blood. Because 2.5 x 10(8) CFU induced a sustained fever without inducing a state of sepsis/severe infection, this dose was used in subsequent experiments. The E. coli-induced increase in bT was preceded by a decrease in Tsk, reflecting a thermoregulatory response. TNF-alpha, IL-1 beta, and IL-6 were detected at 3 h in serum of animals injected i.p. with E. coli. In the peritoneal exudates, TNF-alpha, IL-1 beta, and IL-6 were detected at 0.5 and 3 h after E. coli administration. Moreover, both IL-1 beta and IL-6, but not TNF-alpha, were found in the cerebrospinal fluid (CSF) and hypothalamus of animals injected with E. coli. Although pre-treatment (i.c.v., 2 mu l, 15 min before) with anti-IL-6 antibody (anti-IL-6, 5 mu g) reduced E. coli-induced fever, pre-treatment with either IL-1 receptor antagonist (IL-1ra, 200 mu g) or soluble TNF receptor I (sTNFRI, 500 ng) had no effect on the fever response. In conclusion, replicating E. coli promotes an integrated thermoregulatory response in which the central action of IL-6, but not IL-1 and TNF, appears to be important.