Rosalina Gavín

Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation

Mesophilic Aeromonas strains express a single polar flagellum in all culture conditions and produce lateral flagella on solid media. Such hyperflagellated cells demonstrate increased adherence. Nine lateral flagella genes, lafA–U for Aeromonas hydrophila, and four Aeromonas caviae genes, lafA1, lafA2, lafB and fliU, were isolated. Mutant characterization, nucleotide and N‐terminal sequencing demonstrated that the A. hydrophila and A. caviae lateral flagellins were almost identical, but were distinct from their polar flagellum counterparts. The aeromonad lateral flagellins exhibited higher molecular masses on SDS–PAGE, and this aberrant migration was thought to result from post‐translational modification through glycosylation. Mutation of the Aeromonas lafB, lafS or both A. caviae lateral flagellins caused the loss of lateral flagella and a reduction in adherence and biofilm formation. Mutations in lafA1, lafA2, fliU or lafT resulted in strains that expressed lateral flagella, but had reduced adherence levels. Mutation of the lateral flagella loci did not affect polar flagellum synthesis, but the polarity of the transposon insertions on the A. hydrophila lafT/U genes resulted in non‐motility. However, mutations that abolished polar flagellum production also inhibited lateral flagella expression. We conclude that Aeromonas lateral flagella: (i) play a role in adherence and biofilm formation; (ii) are distinct from the polar flagellum; (iii) synthesis is dependent upon the presence of a polar flagellum filament; and (iv) that the motor proteins of the polar and lateral flagella systems appear to be shared.

Enhanced susceptibility of Prnp-deficient mice to kainate-induced seizures, neuronal apoptosis, and death: Role of AMPA/kainate receptors

Normal physiologic functions of the cellular prion protein (PrPc) are still elusive. This GPI‐anchored protein exerts many functions, including roles in neuron proliferation, neuroprotection or redox homeostasis. There are, however, conflicting data concerning its role in synaptic transmission. Although several studies report that PrPc participates in NMDA‐mediated neurotransmission, parallel studies describe normal behavior of PrPc‐mutant mice. Abnormal axon connections have been described in the dentate gyrus of the hippocampi of PrPc‐deficient mice similar to those observed in epilepsy. A study indicates increased susceptibility to kainate (KA) in these mutant mice. We extend the observation of these studies by means of several histologic and biochemical analyses of KA‐treated mice. PrPc‐deficient mice showed increased sensitivity to KA‐induced seizures in vivo and in vitro in organotypic slices. In addition, we show that this sensitivity is cell‐specific because interference experiments to abolish PrPc expression increased susceptibility to KA in PrPc‐expressing cells. We indicate a correlation of susceptibility to KA in cells lacking PrPc with the differential expression of GluR6 and GluR7 KA receptor subunits using real‐time RT‐PCR methods. These results indicate that PrPc exerts a neuroprotective role against KA‐induced neurotoxicity, probably by regulating the expression of KA receptor subunits.

Increased oxidation, glycoxidation, and lipoxidation of brain proteins in prion disease

The basic molecular underpinnings of the pathological changes that unfold in prion disease remain elusive. A key role of increased oxidative stress has been hypothesized. Given the transient nature of most intermediate molecules implicated, increased oxidative stress is better assessed by quantitating the damage it causes to macromolecules. We used mass spectrometry-based methods to measure specific products of protein oxidation, glycoxidation, and lipoxidation in brains from patients suffering from Creutzfeldt-Jakob disease and Syrian hamsters affected by scrapie. In both cases, increased amounts of glutamic and aminoadipic semialdehydes, products of metal-catalyzed oxidation, malondialdehydelysine (a product of lipoxidation), N-epsilon-carboxyethyllysine (a product of glycoxidation), and N-epsilon-carboxymethyllysine (generated by lipoxidation and glycoxidation) were measured. PrP(Sc), the infectious isoform of the prion protein that accumulates in prion disease, was itself shown to be a target of increased oxidative modification. These changes were accompanied by alterations in fatty acid composition and increased phosphorylation of ERK(1/2) and p38, protein kinases known to respond to increased flows of ROS. These data support an important role of oxidative damage in the pathology of prion disease.

Lateral flagella are required for increased cell adherence, invasion and biofilm formation by Aeromonas spp.

Two types of flagella are responsible for motility in mesophilic Aeromonas strains. A polar unsheathed flagellum is expressed constitutively that allows the bacterium to swim in liquid environments and, in media where the polar flagellum is unable to propel the cell, Aeromonas express peritrichous lateral flagella. Recently, Southern blot analysis using a DNA probe based on the Aeromonas caviae Sch3N lateral flagellin gene sequence showed a good correlation between strains positive for the DNA probe, swarming motility and the presence of lateral flagella by microscopy. Here, we conclude that the easiest method for the detection of the lateral flagellin gene(s) is by PCR (polymerase chain reaction); this showed good correlation with swarming motility and the presence of lateral flagella. This was despite the high degree of DNA heterogeneity found in Aeromonas gene sequences. Furthermore, by reintroducing the laf (lateral flagella) genes into several mesophilic lateral-flagella-negative Aeromonas wild-type strains, we demonstrate that this surface structure enhances the adhesion to and invasion of HEp-2 cells and the capacity for biofilm formation in vitro. These results, together with previous data obtained using Laf- mutants, demonstrate that lateral flagella production is a pathogenic feature due to its enhancement of the interaction with eukaryotic cell surfaces.

Regulation of GABAA and Glutamate Receptor Expression, Synaptic Facilitation and Long-Term Potentiation in the Hippocampus of Prion Mutant Mice

Background Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrPsc) of the natural cellular prion protein (PrPc) encoded by the Prnp gene. Although several roles have been attributed to PrPc, its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrPc studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. Methodology/Principal Findings Here we explore the role of PrPc expression in neurotransmission and neural excitability using wild-type, Prnp −/− and PrPc-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp −/− mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina™ microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp −/− and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABAA and AMPA-kainate receptors are co-regulated in both Prnp −/− and Tg20 mice. Conclusions/Significance Present results demonstrate that PrPc is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABAA and AMPA-Kainate neurotransmission. New PrPc functions have recently been described, which point to PrPc as a target for putative therapies in Alzheimers disease. However, our results indicate that a “gain of function” strategy in Alzheimers disease, or a “loss of function” in prionopathies, may impair PrPc function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies.

Role of flm locus in mesophilic Aeromonas species adherence.

ABSTRACT The adherence mechanism of Aeromonas caviae Sch3N to HEp-2 cells was initially investigated through four mini-Tn5 mutants that showed a 10-fold decrease in adherence. These mutants lost motility, flagella, and their lipopolysaccharide (LPS) O antigen (O-Ag). Three genes,flmB-neuA-flmD, were found to be interrupted by the transposon insertions; additionally, two other genes, one lying upstream (flmA) and one downstream (neuB), were found to be clustered in the same operon. While the flmAand flmB genes were present in all mesophilicAeromonas spp. (A. hydrophila, A. caviae, A. veronii bv. veronii, andA. veronii bv. sobria) tested, this was not the case for the neuA-flmD-neuB genes. Construction and characterization of flmB insertion mutants in five other mesophilic Aeromonas strains revealed the loss of motility, flagella, and adherence but did not alter the LPS composition of these strains. Taking the above findings into consideration, we conclude (i) that flagella and possibly the LPS O-Ag are involved in the adherence of the mesophilic Aeromonas to human epithelial cells; (ii)flmA and flmB are genes widely distributed in the mesophilic Aeromonas and are involved in flagella assembly, and thus adherence; and (iii) in A. caviae Sch3N the flmA and flmB genes are found in a putative operon together with neuA, flmD, andneuB and are involved in LPS O-Ag biosynthesis and probably have a role in flagellum assembly.

New insights into cellular prion protein (PrPc) functions: the "ying and yang" of a relevant protein.

The conversion of cellular prion protein (PrP(c)), a GPI-anchored protein, into a protease-K-resistant and infective form (generally termed PrP(sc)) is mainly responsible for Transmissible Spongiform Encephalopathies (TSEs), characterized by neuronal degeneration and progressive loss of basic brain functions. Although PrP(c) is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully determined. Recent studies have confirmed its participation in basic physiological processes such as cell proliferation and the regulation of cellular homeostasis. Other studies indicate that PrP(c) interacts with several molecules to activate signaling cascades with a high number of cellular effects. To determine PrP(c) functions, transgenic mouse models have been generated in the last decade. In particular, mice lacking specific domains of the PrP(c) protein have revealed the contribution of these domains to neurodegenerative processes. A dual role of PrP(c) has been shown, since most authors report protective roles for this protein while others describe pro-apoptotic functions. In this review, we summarize new findings on PrP(c) functions, especially those related to neural degeneration and cell signaling.

A polar flagella operon (flg) of Aeromonas hydrophila contains genes required for lateral flagella expression.

Aeromonas spp. are pathogens of both humans and poikilothermic animals, causing a variety of diseases. Certain strains are able to produce two distinct types of flagella; polar flagella for swimming in liquid and lateral flagella for swarming over surfaces. Although, both types of flagella have been associated as colonisation factors, little is known about their organisation and expression. Here we characterised a complete flagellar locus of Aeromonas hydrophila (flg) containing 16 genes, this was analogous to region 1 of the Vibrio parahaemolyticus polar flagellum, with the difference that no flagellin genes were found on A. hydrophila while V. parahaemolyticus showed three flagellin genes. The flg region was present in all Aeromonas strain tested. Defined insertion mutants in flgL, were unable to swim, had a drastic reduction in swarming, lateral flagella, HEp-2 cell adhesion and biofilm formation. Mutations in flgN caused a drastic reduction in lateral flagella, inability to swarm, but these strains were still able to swim. Whereas the cheV mutants still produced both types of flagella and were able to swim and swarm. These results suggest that FlgN is required for lateral flagella formation and swarming motility, but not for polar flagellum-mediated swimming.

A Semaphorin 3A Inhibitor Blocks Axonal Chemorepulsion and Enhances Axon Regeneration

Secreted semaphorins are a large group of extracellular proteins involved in a variety of processes during development, including neuronal migration and axon guidance. We screened a peptoid combinatorial library to search for semaphorin 3A inhibitors, and identified a peptoid (SICHI: semaphorin Induced chemorepulsion inhibitor) that blocks semaphorin 3A-chemorepulsion and growth-cone collapse in axons at millimolar concentrations. SICHI inhibits the binding of semaphorin 3A to its receptor complex (neuropilin 1/plexin A1) and semaphorin 3A-induced phosphorylation of GSK3. Chemorepulsion induced by semaphorin 3F or netrin 1 is not blocked by SICHI. We also show that SICHI promotes neural regeneration of damaged axons. We suggest that SICHI, a selective inhibitor of semaphorin 3A, is of therapeutic interest for approaches aimed at promoting axonal regeneration and brain repair.

The inner-core lipopolysaccharide biosynthetic waaE gene: function and genetic distribution among some Enterobacteriaceae

To determine the function of the waaE gene in the biosynthesis of the inner-core LPS of Klebsiella pneumoniae, a waaE non-polar mutant has been constructed. Data obtained from the comparative chemical analysis of LPS samples obtained from the wild-type, the mutant strain and the complemented mutant demonstrated that the waaE gene is involved in substitution of alpha-L-glycero-D-manno-heptopyranose I (L,D-HeppI) at the O-4 position by a beta-D-glucopyranose (beta-D-Glcp) residue. In addition, DNA amplification and nucleotide sequence determination studies revealed that waaE homologues located between the waaA and coaD genes are present in clinical isolates of Enterobacteriaceae containing the structure beta-D-Glcp-(1-->4)-alpha-L,D-HeppI (K. pneumoniae, Proteus mirabilis and Yersinia enterocolitica), as well as in strains of Serratia marcescens and Enterobacter aerogenes of unknown LPS-core structures. Complementation studies using non-polar waaE mutants prove that all the waaE homologues perform the same function. Furthermore, K. pneumoniae, Ser. marcescens and P. mirabilis non-polar waaE mutants showed reduced adhesion and pathogenicity. In addition, the Ser. marcescens and P. murabilis waaE mutants showed reduced swarming motility and ability to form biofilms in vitro. All these characteristics were rescued by reintroduction of the waaE gene independently of its origin. An easy DNA amplification method to detect this gene was established, which also helps in finding the potential presence of this structural feature [beta-D-Glcp-(1-->4)-alpha-L,D-HeppI] in the inner-core LPS of Enterobacteriaceae members with unknown LPS-core structures.