The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa – a review

Pseudomonas aeruginosa (Pa) produces several surface-associated adherence factors or adhesins which promote attachment to epithelial cells and contribute to the virulence of this pathogen. Among them, the type-4 pilus accounts for about 90% of the adherence capability of Pa to human lung pneumocyte A549 cells. Furthermore, it is responsible for more than 90% of the virulence in AB.Y/SnJ mice. Pa type-4 pili display a tip-base differentiation with the adherence function located at the tip of the pilus. All Pa pili prototypes characterized so far contain an intrachain disulfide loop (DSL) of 12 to 17 semi-conserved amino acid residues at the C-terminus of pilin. In Pa, this DSL comprises the epithelial cell-binding domain. Despite little sequence homology, DSL-containing peptides of different pilin prototypes seemingly reveal striking structural similarities. Two β-turns within the loop and the disulfide bridge impose significant structural rigidity on the DSL pilin peptide, suggesting a conformationally conserved binding domain. Insertions of C-terminal pilin peptides with disrupted DSL displayed on the surface of bacterial S-layer mediate the same receptor binding characteristics as pili, indicating that a DSL is not essential in maintaining the functionality of the binding domain. Pa pili bind specifically to the carbohydrate moiety of the glycosphingolipids (GSL) asialo-G_M1 and asialo-G_M2 and, to a much weaker extent, to lactosyl ceramide and ceramide trihexoside. The disaccharide sequence GalNAcβ(1-4)Gal, common in both asialo-G_M1 and asialo-G_M2, likely represents the minimal structural receptor motif recognized by the pili. Pa pili also bind to surface-localized proteins of human epithelial cells and other cell types, suggesting that non-sialylated GSL and (glyco)proteins function as receptors of pili. In addition to the major pilus adhesin, exoenzyme S and, as recent studies indicate, flagella, are further protein adhesins of Pa with GSL receptor binding specificities similar to those of pili.     

Molecular genetic analysis of type-4 pilus biogenesis and twitching motility using Pseudomonas aeruginosa as a model system – a review

Genetic analysis of Pseudomonas aeruginosa pilus biogenesis and twitching motility has revealed the requirement for several pil loci which have been localized to different regions of the chromosome. One pil locus, designated pilE, resides at approx. 71 min on the PAO genetic map, a region of the chromosome previously shown to harbor a number of genes required for pilus assembly (i.e., pilA, -B, -C, -D, -R and -S). The PilE protein shows significant sequence identity to the N-terminal domain of PilA as well as to the pilin precursors from a variety of type-4 pilus producers. Included within this homologous region is a short, positively charged leader sequence followed by a prepilin peptidase cleavage site and a largely hydrophobic region. Additionally, an unlinked set of pil genes, designated pilG, -H, -I, -J and -K, has been localized to the SpeI fragment H which corresponds to approx. 20 min on the PAO genetic map. This gene cluster encodes proteins that demonstrate remarkable similarity to the chemotaxis proteins of enterics and the gliding bacterium Myxococcus xanthus and are thought to be part of a signal transduction system that controls P. aeruginosa pilus biosynthesis and twitching motility.     

Control of fermenters – a review

Fermenter control has been an active area of research and has attracted more attention in recent years. This is due to the new developments in other related areas which can be exploited to overcome the inherent difficulties in fermenter control. Beginning with conventional regulatory control of operating variables such as temperature, pH and dissolved oxygen concentration, research in fermenter control has undergone significant changes including the recent neural network based approaches. The objective of the paper is to focus the attention of the researchers to the developments in the control of batch, fed-batch and continuous fermenters over the past few years.     

Phenogenetic Characterization of a Group of Giant φKZ-like Bacteriophages of Pseudomonas aeruginosa

A comparative study was made of a group ofPseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage KZ genome (288 334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1–19]. By DNA homology, the phages were assigned to three species (represented by phages KZ, Lin68, and EL, respectively) and two new genera (KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the KZ species (KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the KZ genus. The origin and evolution of the KZ-like phages are discussed. Analysis of protein sequences encoded by the phage KZ genome made it possible to assume wide migration of the KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.     

Binding and transport of transforming DNA by Bacillus subtilis: the role of type-IV pilin-like proteins – a review

The pathway for binding, processing and transport of transforming DNA into competent cells of Bacillus subtilis is described. The known proteins involved in mediating these processes are reviewed in turn, including several that resemble proteins required for type-IV pilus assembly and function, and those involved in protein secretion. Based on the phenotypes of null mutations in the cognate genes for these proteins, on similarities to other proteins and on membrane localization and topology data, proposals are made for the roles of the individual proteins in the transformation process. A dynamic model is suggested for the presentation of transforming DNA to the transport apparatus.     

Potential markers of preeclampsia – a review

Preeclampsia is a leading cause of maternal and fetal/neonatal mortality and morbidity worldwide. The early identification of patients with an increased risk for preeclampsia is therefore one of the most important goals in obstetrics. The availability of highly sensitive and specific physiologic and biochemical markers would allow not only the detection of patients at risk but also permit a close surveillance, an exact diagnosis, timely intervention (e.g. lung maturation), as well as simplified recruitment for future studies looking at therapeutic medications and additional prospective markers. Today, several markers may offer the potential to be used, most likely in a combinatory analysis, as predictors or diagnostic tools. We present here the current knowledge on the biology of preeclampsia and review several biochemical markers which may be used to monitor preeclampsia in a future, that, we hope, is not to distant from today.     

Production of pectolytic enzymes – a review

Pectolytic enzymes play an important role in food processing industries and alcoholic beverage industries. These enzymes degrade pectin and reduce the viscosity of the solution so that it can be handled easily. These enzymes are mainly synthesized by plants and microorganisms. Aspergillus niger is used for industrial production of pectolytic enzymes. This fungus produces polygalacturonase, polymethylgalacturonase and pectinlyase. This review mainly concerns with the production of pectolytic enzymes using different carbon sources. It also deals with the effect of operating parameters such as temperature, aeration rate, agitation and type of fermentation on the production of these enzymes.     

Cryopreservation of eukaryotic algae – a review of methodologies

Since pioneering work in the early 1960s, there has been growing interest and numerous experimental investigations into the cryopreservation of algal material. Mostly, these studies relate to the requirement for long term preservation and storage of algal material contained in culture collections or used in the seaweed mariculture industry. The present review deals with techniques used in the cryopreservation of biological samples and their application to both micro- and macroalgae. Methods for the prevention of cell damage and freezing injury during the cooling and low-temperature storage of algal material are discussed with reference to the effect on viability of such variables as cooling rates, final temperatures attained, the use of various types and concentrations of cryoprotectants, thawing rates, and storage times and temperatures. Some consideration is also given to the various methods used for increasing cell viability, including the induction of freezing tolerance. Cryopreservation protocols employed by numerous workers in this field are detailed, and concluding remarks are made on those techniques and conditions providing optimum viability of cryopreserved algae. This revised version was published online in June 2006 with corrections to the Cover Date.     

The hydraulic architecture of Pinaceae – a review

We reviewed the literature to examine the vulnerability to water stress-induced embolism of Pinaceae relative to other conifers and to study the inter-relationships among the main traits involved in the hydraulic function within the Pinaceae. Results showed that Pinaceae (particularly the genus Pinus) are more vulnerable to xylem embolism, and show less variability in this character, than other conifers. Detailed data from 12 populations of Pinaceae (11 species) from three different areas (Piñol and Sala 2000; Martínez-Vilalta and Piñol 2002; Oliveras et al. 2003) was used to study the relationships among hydraulic properties of stems. These included: leaf-to-wood area ratio (A_L:A _W), wood- and leaf-specific hydraulic conductivity (K_W and K_L, respectively), vulnerability to xylem embolism (Ψ_50PLC), carbon isotope composition of needles (δ¹³C) and minimum needle water potential (minimum Ψ_L). Results showed that hydraulic properties tended to be more correlated among each other than with indicators of environmental (precipitation to potential evapotranspiration ratio, P/E) or physiological water stress (minimum Ψ_L). The only exception was an increase of δ¹³C with decreasing minimum Ψ_L and P/E. Overall, A_L:A _W ratio decreased with increasing vulnerability to xylem embolism, and with increasing K_W and K_L (P<0.05). We found a strong positive relationship between carbon isotope composition and the estimated maximum loss of conductivity due to xylem embolism under field conditions, suggesting stronger stomatal control in more vulnerable species with higher levels of native embolism. Overall, results are consistent with a range of drought-avoidance strategies to minimise the gradient of water potential through the xylem, and show that different relationships among traits are possible depending on the scale of study (individual vs. species or populations). The strong interdependence among hydraulic traits implies that no single trait is a sufficient predictor of drought-resistance in Pinaceae. Finally, it is hypothesised that the intrinsically vulnerable xylem of pines may limit their survival under extremely dry conditions.     

Transdermal applications of ethosomes – a detailed review

Skin, the largest organ of the body serves as a potential route of drug delivery for local and systemic effects. However, the outermost layer of skin, the stratum corneum (SC) acts as a tough barrier that prevents penetration of hydrophilic and high molecular weight drugs. Ethosomes are a novel phospholipid vesicular carrier containing high ethanol concentrations and offer improved skin permeability and efficient bioavailability due to their structure and composition. This article gives a review of ethosomes including their compositions, types, mechanism of drug delivery, stability, and safety behaviour. This article also provides a detailed overview of drug delivery applications of ethosomes in various diseases.     

Characterization of the χψ subcomplex of Pseudomonas aeruginosa DNA polymerase III

Background

The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs) or of the target membrane (t-SNARES), which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth.

Results

Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level.

Conclusions

Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions. When considering splice variants as "natural mutants", evidence on modulation of subcellular localization by variation in domain combination can shed further light on targeting determinants. Although further work will be needed to characterize identified variants, our data might open the route to unravel novel molecular partners and mechanisms, accounting for the multiplicity of functions carried out by the different members of the Longin proteins family.     

Multi-drug resistant Pseudomonas aeruginosa: towards a therapeutic dead end?

Pseudomonas aeruginosa is a major hospital-associated pathogen that can cause severe infections, most notably in patients with cystic fibrosis or those hospitalized in intensive care units. In this context, the current increase in incidence of multi-drug resistant (MDR) isolates of P. aeruginosa (MDRPA) raises serious concerns. MDR in P. aeruginosa is defined as the resistance to 3 or 4 of the following antibiotic classes: penicillins/cephalosporins/monobactams, carbapenems, aminoglycosides, and fluoroquinolones. These strains constantly cumulate several resistance mechanisms as a consequence of multiple genetic events, i.e., chromosomal mutations or horizontal transfers of resistance genes. Involved mechanisms may include active efflux, impermeability resulting from porins loss, plasmid-encoded b-lactamases/carbapenemases or aminoglycosides-modifying enzymes, and enzymatic or mutation-associated changes in antibiotics targets. Antibiotic selection pressure represents the leading risk factor for MDRPA acquisition. Colistin (polymyxin E) remains active on virtually all MDRPA isolates, and increasingly appears as the last available option to treat infections caused by these strains. However, the emergence of colistin resistance has been reported in P. aeruginosa, which may announce the spread of pan-resistant strains in a close future.     

The BapF protein from Pseudomonas aeruginosa is a β-peptidyl aminopeptidase

A gene encoding a so far uncharacterized β-peptidyl aminopeptidase from the opportunistic human pathogen Pseudomonas aeruginosa PAO1 was cloned and actively expressed in the heterologue host Escherichia coli. The gene was identified in the genome sequence by its homology to the S58 family of peptidases. The sequence revealed an open reading frame of 1,101 bp with a deduced amino acid sequence of 366 amino acids. The gene was amplified by PCR, ligated into pET22b(+) and was successfully expressed in E. coli BL21 (DE3). It was shown that the enzyme consists of two polypeptides (α- and β-subunit), which are processed from the precursor. The enzyme is specific for N-terminal β-alanyl dipeptides (β-Ala-Xaa). BapF hydrolyses efficiently β-alanine at the N-terminal position, including H-β³hAla-pNA, H–D-β³hAla-pNA and β-Ala-l-His (l-carnosine). d- and l-alaninamide were also hydrolysed by the enzyme.     

Hydrobiology of the Cochin backwater system – a review

On the south west coast of India, there is an extensiveestuarine system of backwaters, of which Vembanad Lake is the largest. The backwaters of Kerala support as much biological productivity and diversity as tropical rain forests. They are responsible for the rich fisheries potential of Kerala. Cochin backwaters situated at the tip of the northern Vembanad lake is a tropical positive estuarine system extending between 9° 40 and 10° 12N and 76° 10' and 76° 30 E with its northern boundary at Azheekode and southern boundary at Thannirmukham bund. The lake has a length of 80 km and the width varies between 500 and 4000 m. A channel, about 450 m wide at Cochin gut and another at Azheekode, make permanent connections with the Arabian Sea. The depth of the estuary varies considerably. While the shipping channels are maintained at a depth of 10–13 m, the major portion of the estuary has a depth range of 2–7 m. Water from two major rivers viz., Periyar and Muvattupuzha drain into this estuary. During south west monsoon, the estuary is virtually converted into a freshwater basin even in areas around barmouth where salt water penetration occurs below 5 m depth only. The major hydrological variable in the Cochin backwaters is salinity, similar to the situations encountered in estuaries with a gradual declension of salinity from 30 at the entrance of the estuary to 0.2 at the point of entry of the rivers. Salinity gradient in the Cochin backwaters supports diverse species of flora and fauna depending on their capacity to tolerate oligohaline, mesohaline or marine conditions. Low lying swamps and tidal creeks, dominated by sparse patches of mangroves with their nutrient rich physical environment, support larvae and juveniles of many economically important species. Backwaters also act as nursery grounds of commercially important prawns and fishes. The fields around the backwater are suitable for aquaculture. These areas support traditional, seasonal and perennial prawn fishery. The changes in the hydrology controlled by the seasons play an important role in regulating the migrant fauna of the estuary. The Cochin backwater supports a well established endemic fauna. The nutrients and pollutants introduced into the estuary control to a great extent the distribution and abundance of less tolerant species in ecologically sensitive areas in the backwaters. Cochin backwaters, widely regarded as one of the polluted estuaries in India, receive contaminated freshwater inputs and discharges of effluents and partially treated sewage from many points throughout its tidally mixed zone. Recently, changes brought about in the estuary like reclamation and consequent shrinkage of the backwaters and the discharge of pollutants have made an adverse impact on the potential of aquatic ecosystems that used to support high levels of bioproductivity and biodiversity. The construction of Thannirmukham bund and Thottapally spillway to prevent salt water penetration into the paddy fields during pre-monsoon has led to serious ecological problems by interrupting the natural ebb and flow of tides. The hydrography, floral and faunal composition – its spatial and temporal variation plus assessments of the impact of the anthropogenic activities are presented in this review. An attempt to critically evaluate the status of the estuary from the biological and pollutional stand point is also done.     

Analyses of Pseudomonas aeruginosa Lectin Binding to α-Galactosylated Glycans

The specificity and binding capacity of the galactophilic lectin from the Gram negative bacterium Pseudomonas aeruginosa (PA-IL) was determined by solid phase measurements using galactosylated neoglycoproteins immobilized on microtiter plates. The bacterial lectin reacted with both short chain (monosaccharide) and long chain (pentasaccharide) glycoconjugates. Among the Galα1-XGal disaccharides, the highest affinity was observed towards the Galα1-3Gal structure. Raising the incubation temperature enhanced the lectin-polysaccharide agglutination, and it is suggested that binding to certain conformations of polysaccharides could vary between lectins with the same monocarbohydrate specificity and that this activity may, in part, be temperature dependent. Histochemical examination of lectin binding to different porcine tissues suggests a differential glycosylation of the carbohydrate antigens on endothelial cells in various parts of the vascular system. In the pancreas, PA-IL also adhered to the excretory ducts. These observations on PA-IL binding could be of importance both to determine infection foci in P. aeruginosa-mediated vacuities and to determine its role for pancreatic involvement in cystic fibrosis.     

Accuracy of body mass estimates of formalin-preserved fish – a review

This paper highlights possible effects of physical and chemical mechanisms of formalin fixation and preservation on biological tissue and reviews the consequent potential inaccuracies on estimates of body mass of small fishes fixed and preserved in formalin. Twenty-six papers including 65 independent experiments with 35 species which examine effects of formalin on body mass estimates on small fishes are included. The effect of the formalin on the specimens depends on the salinity of the water used to dilute the commercial formalin (usually 1:9 formalin: water) before being used to fixate and preserve fish. Mean wet body mass of the specimens from the studies using seawater or fresh water diluted formalin deceases by 13% and increases by 7%, respectively, from before to after being immersed in formalin. The same trend is found with condition factor in the few papers that report this parameter. Body length decreases on average by c. 2% in fixated and preserved fish regardless of whether the formalin is diluted in seawater or fresh water.     

The Pseudomonas aeruginosa membranes: a target for a new amphiphilic aminoglycoside derivative?

Aminoglycosides are among the most potent antimicrobials to eradicate Pseudomonas aeruginosa. However, the emergence of resistance has clearly led to a shortage of treatment options, especially for critically ill patients. In the search for new antibiotics, we have synthesized derivatives of the small aminoglycoside, neamine. The amphiphilic aminoglycoside 3',4',6-tri-2-naphtylmethylene neamine (3',4',6-tri-2NM neamine) has appeared to be active against sensitive and resistant P. aeruginosa strains as well as Staphylococcus aureus strains (Baussanne et al., 2010). To understand the molecular mechanism involved, we determined the ability of 3',4',6-tri-2NM neamine to alter the protein synthesis and to interact with the bacterial membranes of P. aeruginosa or models mimicking these membranes. Using atomic force microscopy, we observed a decrease of P. aeruginosa cell thickness. In models of bacterial lipid membranes, we showed a lipid membrane permeabilization in agreement with the deep insertion of 3',4',6-tri-2NM neamine within lipid bilayer as predicted by modeling. This new amphiphilic aminoglycoside bound to lipopolysaccharides and induced P. aeruginosa membrane depolarization. All these effects were compared to those obtained with neamine, the disubstituted neamine derivative (3',6-di-2NM neamine), conventional aminoglycosides (neomycin B and gentamicin) as well as to compounds acting on lipid bilayers like colistin and chlorhexidine. All together, the data showed that naphthylmethyl neamine derivatives target the membrane of P. aeruginosa. This should offer promising prospects in the search for new antibacterials against drug- or biocide-resistant strains.     

Does Streptococcus mitis,a neonatal oropharyngeal bacterium,influence the pathogenicity of Pseudomonas aeruginosa?

Microorganisms in a biofilm might promote or suppress each other. We previously found that Pseudomonas aeruginosa (P. aeruginosa) and the normal colonized bacteria in the oropharynx, Streptococcus mitis (S. mitis), were the most common bacteria in the biofilm found on newborns' endotracheal tubes. Here, we found that S. mitis enhanced the adhesion and biofilm formation of P. aeruginosa. Furthermore, it alleviated the immune response induced by P. aeruginosa. These findings remind us that we should not ignore the role of traditionally viewed non-pathogenic bacteria in biofilms and provide new insights into exploring bacterial interaction mechanisms in biofilm related infections.     

A Carbapenem-Resistant Pseudomonas aeruginosa Isolate Harboring Two Copies of blaIMP-34 Encoding a Metallo-β-Lactamase

A carbapenem-resistant strain of Pseudomonas aeruginosa, NCGM1984, was isolated in 2012 from a hospitalized patient in Japan. Immunochromatographic assay showed that the isolate was positive for IMP-type metallo-β-lactamase. Complete genome sequencing revealed that NCGM1984 harbored two copies of bla_IMP-34, located at different sites on the chromosome. Each bla_IMP-34 was present in the same structures of the class 1 integrons, tnpA(ISPa7)-intI1-qacG-bla_IMP-34-aac(6'')-Ib-qacEdelta1-sul1-orf5-tniBdelta-tniA. The isolate belonged to multilocus sequence typing ST235, one of the international high-risk clones. IMP-34, with an amino acid substitution (Glu126Gly) compared with IMP-1, hydrolyzed all β-lactamases tested except aztreonam, and its catalytic activities were similar to IMP-1. This is the first report of a clinical isolate of an IMP-34-producing P. aeruginosa harboring two copies of bla_IMP-34 on its chromosome.