Contribution of bacterial outer membrane vesicles to innate bacterial defense

Background

Microorganisms produce cell-wall-degrading enzymes as part of their strategies for plant invasion/nutrition. Among these, pectin lyases (PNLs) catalyze the depolymerization of esterified pectin by a β-elimination mechanism. PNLs are grouped together with pectate lyases (PL) in Family 1 of the polysaccharide lyases, as they share a conserved structure in a parallel β-helix. The best-characterized fungal pectin lyases are obtained from saprophytic/opportunistic fungi in the genera Aspergillus and Penicillium and from some pathogens such as Colletotrichum gloeosporioides. The organism used in the present study, Colletotrichum lindemuthianum, is a phytopathogenic fungus that can be subdivided into different physiological races with different capacities to infect its host, Phaseolus vulgaris. These include the non-pathogenic and pathogenic strains known as races 0 and 1472, respectively.

Results

Here we report the isolation and sequence analysis of the Clpnl2 gene, which encodes the pectin lyase 2 of C. lindemuthianum, and its expression in pathogenic and non-pathogenic races of C. lindemuthianum grown on different carbon sources. In addition, we performed a phylogenetic analysis of the deduced amino acid sequence of Clpnl2 based on reported sequences of PNLs from other sources and compared the three-dimensional structure of Clpnl2, as predicted by homology modeling, with those of other organisms. Both analyses revealed an early separation of bacterial pectin lyases from those found in fungi and oomycetes. Furthermore, two groups could be distinguished among the enzymes from fungi and oomycetes: one comprising enzymes from mostly saprophytic/opportunistic fungi and the other formed mainly by enzymes from pathogenic fungi and oomycetes. Clpnl2 was found in the latter group and was grouped together with the pectin lyase from C. gloeosporioides.

Conclusions

The Clpnl2 gene of C. lindemuthianum shares the characteristic elements of genes coding for pectin lyases. A time-course analysis revealed significant differences between the two fungal races in terms of the expression of Clpnl2 encoding for pectin lyase 2. According to the results, pectin lyases from bacteria and fungi separated early during evolution. Likewise, the enzymes from fungi and oomycetes diverged in accordance with their differing lifestyles. It is possible that the diversity and nature of the assimilatory carbon substrates processed by these organisms played a determinant role in this phenomenon.     

A bacterial actin unites to divide bacterial cells

EMBO J 31 10, 2249–2260 (2012); published online March302012Once thought to exist only in eukaryotic cells, the highly conserved bacterial cytoskeleton is now known to function analogously to its eukaryotic counterparts, particularly in cell shape and division. For instance, the actin-like MreB protein and its homologs are important to maintain cell shape in many rod-shaped bacteria, probably by organizing how peptidoglycan is synthesized. FtsZ, a tubulin homolog, forms a scaffold for the cytokinetic ring, or divisome, by GTP-dependent polymerization into protofilaments. In this issue of The EMBO Journal, Szwedziak et al (2012) reveal the first crystal structures of cell division protein FtsA polymerizing into actin-like filaments, along with in vivo evidence that this self-interaction is crucial for proper cell division.FtsA is an actin homolog required for cytokinesis in many bacterial species and has several key roles in cell division, including helping to tether FtsZ to the cytoplasmic membrane via a membrane-targeting sequence (MTS), recruiting other essential proteins to the divisome, and perhaps promoting divisome constriction (de Boer, 2010). Szwedziak et al (2012) recapitulate the FtsZ-FtsA-membrane association in vitro using liposomes with FtsZ and FtsA proteins from Thermotoga maritima. To get a closer look at the FtsA-FtsZ interface, the authors co-crystallize FtsA with the carboxy-terminal tail of FtsZ, which is known to interact with FtsA. Intriguingly, the crystal reveals an FtsA homodimer. Contrary to the previous bioinformatics model of FtsA self-interaction that proposed a 180° rotation between the two subunits (Carettoni et al, 2003), the FtsA-FtsA interface in the crystal structure shows no rotation, similar to F-actin. Szwedziak et al (2012) also show that FtsA can form longer, actin-like polymers in the presence of non-hydrolysable ATP or on lipid monolayers. These results are surprising because FtsA has a divergent subdomain architecture compared to other actin-family proteins (van den Ent and Löwe).A critical question now is whether FtsA needs to form polymers in vivo to function properly. Purified Streptococcus pneumoniae FtsA assembles into large polymers that are not like F-actin, and it remains unclear if these structures are relevant in vivo (Krupka et al, 2012). Wild-type FtsA proteins do not form detectable filaments in cells, but C-terminal truncations of FtsA that remove the MTS form polymers quite readily in cells when overproduced, although they are not functional (Pichoff and Lutkenhaus, 2007). Even so, starting with an MTS truncation derivative of FtsA to visualize in vivo polymers, Szwedziak et al (2012) design site-directed mutants of Bacillus subtilis FtsA based on the FtsA-FtsA interface of their crystals; these fail to assemble into polymers in vivo. Using a similar MTS truncation derivative, Pichoff et al (2012) created random mutations in Escherichia coli FtsA, and found that those mapping to the same interface found by Szwedziak et al (2012) also disrupted polymer formation. Together, these data suggest that these residues are needed for FtsA self-interaction. Perplexingly, when these mutants were subsequently tested for functionality in the context of full-length FtsA, the results were mixed. Pichoff et al (2012) showed that FtsA mutants deficient for self-interaction in E. coli have a gain-of-function phenotype, whereas Szwedziak et al (2012) report that analogous mutants in B. subtilis FtsA suffer a loss of function. These results support the idea that FtsA self-association is related to its activity (Shiomi and Margolin, 2007), yet understanding how self-interaction regulates FtsA function clearly requires further study.The ability of eukaryotic cytoskeletal proteins to form long polymers is essential to their function, but the physiological relevance of long polymer formation by bacterial cytoskeletal proteins is now a topic of debate (Figure 1). For example, it has been hypothesized that FtsZ protofilaments wrap around the entire circumference of the cell to form the cytokinetic ring. However, recent studies using photoactivated localization microscopy (PALM) and electron cryotomography reveal a different model in which FtsZ forms a series of very short polymers that overlap to encompass the diameter of the cell (Li et al, 2007; Fu et al, 2010). MreB was also originally thought to form long-range helical polymers extending the length of the cell, but recent data obtained with more sophisticated microscopic techniques suggest that MreB is distributed in patches that move circumferentially and independently (White and Gober, 2012). It is not yet clear which of these models represents the true cellular architecture of MreB, although it is likely that some degree of MreB polymerization is still needed for function. It is notable that other bacterial homologs of actin and tubulin used for generating scaffolds or partitioning plasmid DNA, but not for essential cellular processes such as cell division and growth, tend to form long polymers that extend throughout the cell (Pogliano, 2008). The continued combined use of microscopic, biochemical, and genetic methods, as demonstrated by Szwedziak et al (2012) will enhance future understanding of ancestral tubulin and actin proteins in prokaryotes.Open in a separate windowFigure 1Bacterial actin and tubulin filaments involved in cell growth and division. (A) MreB (purple) has long been thought of as a spiral filament twisting along the cell length to control cell shape. Likewise, FtsZ protofilaments (blue) were once thought to wrap around the cell midpoint to organize the divisome. (B) Recent work using high-resolution microscopy has revealed that long cytoskeletal filaments are more likely to be short patches of polymers. The present work by Szwedziak et al (2012) has added FtsA actin-like filaments (green) to the model of possible divisome architecture.     

Flavin binding by bacterial luciferase: Affinity chromatography of bacterial luciferase

Immobilized FMN covalently attached to Sepharose-6B-hexanoate binds bacterial luciferase. Immobilized flavin is also effective in its reduced form as a substrate in the light emitting reaction catalyzed by luciferase.     

Protection of mice against bacterial infection by oral administration of bacterial lipopolysaccharide

The effect of orally administered bacterial lipopolysaccharide (LPS) on host resistance against bacterial infections was studied. LPS orally given for 5 consecutive days prior to infection caused no apparent toxic effect and protected mice against Pseudomonas aeruginosa and Listeria monocytogenes infections.     

Evolution of bacterial genomes

This review examines evolution of bacterial genomes with an emphasis on RNA based life, the transition to functional DNA and small evolving genomes (possibly plasmids) that led to larger, functional bacterial genomes.     

Biodegradability of bacterial surfactants

This work aimed at evaluating the biodegradability of different bacterial surfactants in liquid medium and in soil microcosms. The biodegradability of biosurfactants by pure and mixed bacterial cultures was evaluated through CO₂ evolution. Three bacterial strains, Acinetobacter baumanni LBBMA ES11, Acinetobacter haemolyticus LBBMA 53 and Pseudomonas sp. LBBMA 101B, used the biosurfactants produced by Bacillus sp. LBBMA 111A (mixed lipopeptide), Bacillus subtilis LBBMA 155 (lipopeptide), Flavobacterium sp. LBBMA 168 (mixture of flavolipids), Dietzia Maris LBBMA 191(glycolipid) and Arthrobacter oxydans LBBMA 201(lipopeptide) as carbon sources in minimal medium. The synthetic surfactant sodium dodecyl sulfate (SDS) was also mineralized by these microorganisms, but at a lower rate. CO₂ emitted by a mixed bacterial culture in soil microcosms with biosurfactants was higher than in the microcosm containing SDS. Biosurfactant mineralization in soil was confirmed by the increase in surface tension of the soil aqueous extracts after incubation with the mixed bacterial culture. It can be concluded that, in terms of biodegradability and environmental security, these compounds are more suitable for applications in remediation technologies in comparison to synthetic surfactants. However, more information is needed on structure of biosurfactants, their interaction with soil and contaminants and scale up and cost for biosurfactant production.     

Phagocytosis of bacterial pathogens

Phagocytosis is an evolutionarily ancient, receptor-driven process, by which phagocytic cells recognize invading microbes and destroy them after internalization. The phagocytosis receptor Eater is expressed exclusively on Drosophila phagocytes and is required for the survival of bacterial infections. In a recent study, we explored how Eater can defend fruit flies against different kinds of bacteria. We discovered that Eater bound to certain types of bacteria directly, while for others bacterial binding was dependent on prior disruption of the bacterial envelope. Similar to phagocytes, antimicrobial peptides and lysozymes are ancient components of animal immune systems. Our results suggest that cationic antimicrobial peptides, as well as lysozymes, can facilitate Eater binding to live Gram-negative bacteria. Both types of molecules promote surface-exposure of bacterial ligands that otherwise would remain buried and hidden under an outer membrane. We propose that unmasking ligands for phagocytic receptors may be a conserved mechanism operating in many animals, including humans. Thus, studying a Drosophila phagocytosis receptor may advance our understanding of innate immunity in general.     

Structure of bacterial lipopolysaccharides

Bacterial lipopolysaccharides are the major components of the outer surface of Gram-negative bacteria They are often of interest in medicine for their immunomodulatory properties. In small amounts they can be beneficial, but in larger amounts they may cause endotoxic shock. Although they share a common architecture, their structural details exert a strong influence on their activity. These molecules comprise: a lipid moiety, called lipid A, which is considered to be the endotoxic component, a glycosidic part consisting of a core of approximately 10 monosaccharides and, in "smooth-type" lipopolysaccharides, a third region, named O-chain, consisting of repetitive subunits of one to eight monosaccharides responsible for much of the immunospecificity of the bacterial cell.     

DOLOP--database of bacterial lipoproteins

Bacterial lipoproteins and lipid modification are gaining importance owing to their essential nature, roles in pathogenesis and interesting commercial applications. We have created an exclusive knowledge base for bacterial lipoproteins by processing information from 510 entries to provide a list of 199 distinct lipoproteins with relevant links to molecular details. Features include functional classification, predictive algorithm for query sequences, primary sequence analysis and lists of predicted lipoproteins from 43 completed bacterial genomes along with interactive information exchange facility. AVAILABILITY: The website called Database Of bacterial LipOProteins (DOLOP) is available at http://www.mrc-lmb.cam.ac.uk/genomes/dolop along with additional information on the biosynthetic pathway, supplementary material and other related figures.     

Phagocytosis of bacterial pathogens

Phagocytosis is an evolutionarily ancient, receptor-driven process, by which phagocytic cells recognize invading microbes and destroy them after internalization. The phagocytosis receptor Eater is expressed exclusively on Drosophila phagocytes and is required for the survival of bacterial infections. In a recent study, we explored how Eater can defend fruit flies against different kinds of bacteria. We discovered that Eater bound to certain types of bacteria directly, while for others bacterial binding was dependent on prior disruption of the bacterial envelope. Similar to phagocytes, antimicrobial peptides and lysozymes are ancient components of animal immune systems. Our results suggest that cationic antimicrobial peptides, as well as lysozymes, can facilitate Eater binding to live Gram-negative bacteria. Both types of molecules promote surface-exposure of bacterial ligands that otherwise would remain buried and hidden under an outer membrane. We propose that unmasking ligands for phagocytic receptors may be a conserved mechanism operating in many animals, including humans. Thus, studying a Drosophila phagocytosis receptor may advance our understanding of innate immunity in general.