Pathogenic LRRK2 mutations do not alter gene expression in cell model systems or human brain tissue

Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances.

An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5 alpha and EQ1

Aims: To examine Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 for known pathogenic mechanisms. Methods and Results: Using specific DNA probes, the strains were shown not to carry the genes encoding invasion, various adhesion phenotypes or expression of a range of enterotoxins. The strains were unable to express long-chain lipopolysaccharide and were susceptible to the effects of serum complement. Using a BALB/c mouse model, the strains were shown to be unable to survive in selected tissues or to persist in the mouse gut. Using a chick model, strains EQ1, BLR and BL21 invaded livers but not spleens; only strain EQ1 persisted in the chick gut. In Merino sheep, only strain EQ1 was detected 6 d postinfection. Conclusions: Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 did not carry the well-recognized pathogenic mechanisms required by strains of E. coli causing the majority of enteric infections. Significance and Impact of the Study: Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 were considered to be non-pathogenic and unlikely to survive in host tissues and cause disease.

Variation in genome organization of the plant pathogenic fungus Colletotrichum lindemuthianum

The genome structure of Colletotrichum lindemuthianum in a set of diverse isolates was investigated using a combination of physical and molecular approaches. Flow cytometric measurement of genome size revealed significant variation between strains, with the smallest genome representing 59% of the largest. Southern-blot profiles of a cloned fungal telomere revealed a total chromosome number varying from 9 to 12. Chromosome separations using pulsed-field gel electrophoresis (PFGE) showed that these chromosomes belong to two distinct size classes: a variable number of small (< 2.5 Mb) polymorphic chromosomes and a set of unresolved chromosomes larger than 7 Mb. Two dispersed repeat elements were shown to cluster on distinct polymorphic minichromosomes. Single-copy flanking sequences from these repeat-containing clones specifically marked distinct small chromosomes. These markers were absent in some strains, indicating that part of the observed variability in genome organization may be explained by the presence or absence, in a given strain, of dispensable genomic regions and/or chromosomes.

Pathogenic Parkinson’s disease mutations across the functional domains of LRRK2 alter the autophagic/lysosomal response to starvation

LRRK2 is one of the most important genetic contributors to Parkinson’s disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consis- tently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data high- light the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations.

OCCURRENCE OF PATHOGENIC MICROORGANISMS IN FISH SOLD IN SAO PAULO, BRAZIL

This study investigated the presence of potentially human pathogenic strains of Vibrio spp., Aeromonas spp., Escherichia coli, Salmonella spp. and Staphylococcus aureus in fish commercialized in street markets of Sao Paulo city, Brazil. Twenty fish of different species were analyzed for foodborne pathogens using conventional methods. High levels of fecal contamination were detected in 25% of samples. S. aureus was isolated from 10% of samples. All were negative for Salmonella. Vibrio species, including Vibrio cholerae non-O1/non-O139, were observed in 85% of samples although Vibrio parahaemolyticus was not found in this study. Aeromonas spp., including A. hydrophila, was isolated from 50% of fish samples. The occurrence of these pathogens suggests that the fish commercialized in Sao Paulo may represent a health risk to the consumers.

Genotyping, physiological features and proteolytic activities of a potentially pathogenic Acanthamoeba sp isolated from tap water in Brazil

Acanthamoeba spp., known to cause keratitis and granulomatous encephalitis in humans, are frequently isolated from a variety of water sources. Here we report for the first time the characterization of an Acanthamoeba sp. (ACC01) isolated from tap water in Brazil. This organism is currently being maintained in an axenic growth medium. Phylogenetic analysis based on SSU rRNA gene sequences positioned the new isolate in genotype T4, closest to the keratitis-causing isolate, A. polyphaga ATCC 30461 (similar to 99% similarity). Acanthamoeba ACC01 and A. polyphaga 30461 both grew at 37 degrees C and were osmotically resistant, multiplying in hyperosmolar medium. Both isolates secreted comparable amounts of proteolytic enzymes, including serine peptidases that were optimally active at a near neutral/alkaline pH and resolved identically in gelatin gels. Incubation of gels at pH 4.0 with 2 mM DTT also indicated the secretion of similar cysteine peptidases. Altogether, the results point to the pathogenic potential of Acanthamoeba ACC01. (C) 2009 Elsevier Inc. All rights reserved.

Melanin as a virulence factor of Paracoccidioides brasiliensis and other dimorphic pathogenic fungi: a minireview

Melanin pigments are substances produced by a broad variety of pathogenic microorganisms, including bacteria, fungi, and helminths. Microbes predominantly produce melanin pigment via tyrosinases, laccases, catecholases, and the polyketide synthase pathway. In fungi, melanin is deposited in the cell wall and cytoplasm, and melanin particles (""ghosts"") can be isolated from these fungi that have the same size and shape of the original cells. Melanin has been reported in several human pathogenic dimorphic fungi including Paracoccidioides brasiliensis, Sporothrix schenckii, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides posadasii. Melanization appears to contribute to virulence by reducing the susceptibility of melanized fungi to host defense mechanisms and antifungal drugs.

Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Leptospixosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-angstrom-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL32(21-272)). The LipL32(21-272) monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL32(21-272) is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL32(21-272). Ca(2+) binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca(2+) as no structural or stability perturbations were observed for Mg(2+), Zn(2+), or Cu(2+). Careful examination of the crystal lographic structure suggests the locations of putative regions that could mediate Ca(2+) binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and lamixidn. (C) 2009 Elsevier Ltd. All rights reserved.

Biosurfactants as Agents to Reduce Adhesion of Pathogenic Bacteria to Polystyrene Surfaces: Effect of Temperature and Hydrophobicity

Polystyrene surfaces were conditioned with surfactin and rhamnolipid biosurfactants and then assessed regarding the attachment of Staphylococcus aureus, Listeria monocytogenes, and Micrococcus lute us. The effect of different temperatures (35, 25, and 4 degrees C) on the anti-adhesive activity was also studied. Microbial adhesion to solvents and contact angle measurements were performed to characterize bacteria and material surfaces. The results showed that surfactin was able to inhibit bacterial adhesion in all the conditions analyzed, giving a 63-66% adhesion reduction in the bacterial strains at 4 degrees C. Rhamnolipid promoted a slight decrease in the attachment of S. aureus. The anti-adhesive activity of surfactin increased with the decrease in temperature, showing that this is an important parameter to be considered in surface conditioning tests. Surfactin showed good potential as an anti-adhesive compound that can be explored to protect surfaces from microbial contamination.

Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces

Aims: To investigate the effect of the biosurfactants surfactin and rhamnolipids on the adhesion of the food pathogens Listeria monocytogenes, Enterobacter sakazakii and Salmonella Enteritidis to stainless steel and polypropylene surfaces. Methods and Results: Quantification of bacterial adhesion was performed using the crystal violet staining technique. Preconditioning of surfaces with surfactin caused a reduction on the number of adhered cells of Ent. sakazakii and L. monocytogenes on stainless steel. The most significant result was obtained with L. monocytogenes where number of adhered cells was reduced by 10(2) CFU cm(-2). On polypropylene, surfactin showed a significant decrease on the adhesion of all strains. The adsorption of surfactin on polystyrene also reduces the adhesion of L. monocytogenes and Salm. Enteritidis growing cells. For short contact periods using nongrowing cells or longer contact periods with growing cells, surfactin was able to delay bacterial adhesion. Conclusions: The prior adsorption of surfactin to solid surfaces contributes on reducing colonization of the pathogenic bacteria. Significance and Impact of the Study: This is the first work investigating the effect of surfactin on the adhesion of the food pathogens L. monocytogenes, Ent. sakazakii and Salm. Enteritidis to polypropylene and stainless steel surfaces.

The Isolation and Characterization of Multiply Antibiotic Resistant Strains of Fish Pathogenic Flavobacterium Species

Since the development of the first antibiotics in the 1940’s, there has been widespread overuse in both clinical and agricultural applications. Antibiotic resistance has become a significant problem as a result of subsequent dissemination of antibiotics into the environment, and multiply-resistant strains of bacteria are now a major pathogenic threat. In this study eight separate strains of Flavobacterium responsible for recent disease outbreaks in fish hatcheries throughout Maine were collected and analyzed. All eight strains were found to be resistant to high levels of a number of different antibiotics, including those used for aquaculture as well as human chemotherapeutic applications. Flavobacterium isolates were also shown phenotypically to transfer antibiotic resistance determinants using a conjugation mating system in which Flavobacterium was the donor and Escherichia coli DH5- alpha was the recipient. This experiment suggests that it may be possible for Flavobacterium strains to transfer their multiple antibiotic resistance determinants to human pathogenic bacterial strains. Importantly, none of the hatcheries from which the Flavobacterium isolates were obtained had ever used antibiotics to treat their fish stock. It is possible that there is another selective agent responsible for the development of antibiotic resistance in the absence of antibiotic pressure. Mercury is one possible candidate, as all of the strains tested were resistant to mercuric chloride and it is known that genes encoding antibiotic resistance can be carried on the same mobile genetic elements that encode for mercury resistance. Preliminary data also suggest that the majority of the Flavobacterium isolates contain genes for mercuric ion reduction, which would confirm the mercury resistance genotype.

Serological investigation and PCR in detection of pathogenic leptospires in snakes

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

Isolation of extraintestinal pathogenic Escherichia coli from diarrheic dogs and their antimicrobial resistance profile

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

Protection levels of vaccinated pigeons (Columba livia) against a highly pathogenic newcastle disease virus strain

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