Genome-Wide Analyses of the Temperature-Responsive Genetic Loci of the Pectinolytic Plant Pathogenic Pectobacterium atrosepticum

Temperature is one of the critical factors affecting gene expression in bacteria. Despite the general interest in the link between bacterial phenotypes and environmental temperature, little is known about temperature-dependent gene expression in plant pathogenic Pectobacterium atrosepticum, a causative agent of potato blackleg and tuber soft rot worldwide. In this study, twenty-nine P. atrosepticum SCRI1043 thermoregulated genes were identified using Tn5-based transposon mutagenesis coupled with an inducible promotorless gusA gene as a reporter. From the pool of 29 genes, 14 were up-regulated at 18 °C, whereas 15 other genes were up-regulated at 28 °C. Among the thermoregulated loci, genes involved in primary bacterial metabolism, membrane-related proteins, fitness-corresponding factors, and several hypothetical proteins were found. The Tn5 mutants were tested for their pathogenicity in planta and for features that are likely to remain important for the pathogen to succeed in the (plant) environment. Five Tn5 mutants expressed visible phenotypes differentiating these mutants from the phenotype of the SCRI1043 wild-type strain. The gene disruptions in the Tn5 transposon mutants caused alterations in bacterial generation time, ability to form a biofilm, production of lipopolysaccharides, and virulence on potato tuber slices. The consequences of environmental temperature on the ability of P. atrosepticum to cause disease symptoms in potato are discussed.     

Role of Aeromonas hydrophila Flagella Glycosylation in Adhesion to Hep-2 Cells,Biofilm Formation and Immune Stimulation

Abstract: Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous heptasaccharide glycan. Two mutants with altered (light and strong) polar flagella glycosylation still able to produce flagella were previously obtained, as well as mutants lacking the O34-antigen lipopolysaccharide (LPS) but with unaltered polar flagella glycosylation. We compared these mutants, altogether with the wild type strain, in different studies to conclude that polar flagella glycosylation is extremely important for A. hydrophila adhesion to Hep-2 cells and biofilm formation. Furthermore, the polar flagella glycosylation is an important factor for the immune stimulation of IL-8 production via toll receptor 5 (TLR5).     

Pectobacterium parmentieri SCC 3193 Mutants with Altered Synthesis of Cell Surface Polysaccharides Are Resistant to N4-Like Lytic Bacteriophage ϕA38 (vB_Ppp_A38) but Express Decreased Virulence in Potato (Solanum tuberosum L.) Plants

Pectobacterium parmentieri is a Gram-negative plant-pathogenic bacterium able to infect potato (Solanum tuberosum L.). Little is known about lytic bacteriophages infecting P. parmentieri and how phage-resistance influences the environmental fitness and virulence of this species. A lytic phage vB_Ppp_A38 (ϕA38) has been previously isolated and characterized as a potential biological control agent for the management of P. parmentieri. In this study, seven P. parmentieri SCC 3193 Tn5 mutants were identified that exhibited resistance to infection caused by vB_Ppp_A38 (ϕA38). The genes disrupted in these seven mutants encoded proteins involved in the assembly of O-antigen, sugar metabolism, and the production of bacterial capsule exopolysaccharides. The potential of A38-resistant P. parmentieri mutants for plant colonization and pathogenicity as well as other phenotypes expected to contribute to the ecological fitness of P. parmentieri, including growth rate, use of carbon and nitrogen sources, production of pectinolytic enzymes, proteases, cellulases, and siderophores, swimming and swarming motility, presence of capsule and flagella as well as the ability to form biofilm were assessed. Compared to the wild-type P. parmentieri strain, all phage-resistant mutants exhibited a reduced ability to colonize and to cause symptoms in growing potato (S. tuberosum L.) plants. The implications of bacteriophage resistance on the ecological fitness of P. parmentieri are discussed.     

Virulence Factors of Erwinia amylovora: A Review

Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3′-5′)-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them.     

Isolation of the Autoinducer-Quenching Strain that Inhibits LasR in Pseudomonas aeruginosa

Quorum sensing (QS) has been recognized as a general phenomenon in microorganisms and plays an important role in many pathogenic bacteria. In this report, we used the Agrobacterium tumefaciens biosensor strain NT1 to rapidly screen for autoinducer-quenching inhibitors from bacteria. After initial screening 5389 isolates obtained from land and beach soil, 53 putative positive strains were identified. A confirmatory bioassay was carried out after concentrating the putative positive culture supernatant, and 22 strains were confirmed to have anti-LasR activity. Finally, we determined the strain JM2, which could completely inhibit biofilm formation of Pseudomonas aeruginosa PAO1, belonged to the genus Pseudomonas by analysis of 16S rDNA. Partially purified inhibitor factor(s) F5 derived from culture supernatants specifically inhibited LasR-controlled elastase and protease in wild type P. aeruginosa PAO1 by 68% and 73%, respectively, without significantly affecting growth; the rhl-controlled pyocyanin and rhamnolipids were inhibited by 54% and 52% in the presence of 100 μg/mL of F5. The swarming motility and biofilm of PAO1 were also inhibited by F5. Real time RT-PCR on samples from 100 μg/mL F5-treated P. aeruginosa showed downregulation of autoinducer synthase (LasRI and rhlI) and cognate receptor (lasR and rhlR) genes by 50%, 28%, 48%, and 29%, respectively. These results provide compelling evidence that the F5 inhibitor(s) interferes with the las system and significantly inhibits biofilm formation.     

Inhibition of Virulence Factors and Biofilm Formation by Wogonin Attenuates Pathogenicity of Pseudomonas aeruginosa PAO1 via Targeting pqs Quorum-Sensing System

Pseudomonas aeruginosa, an important opportunistic pathogen, is capable of producing various virulence factors and forming biofilm that are regulated by quorum sensing (QS). It is known that targeting virulence factor production and biofilm formation instead of exerting selective pressure on growth such as conventional antibiotics can reduce multidrug resistance in bacteria. Therefore, many quorum-sensing inhibitors (QSIs) have been developed to prevent or treat this bacterial infection. In this study, wogonin, as an active ingredient from Agrimonia pilosa, was found to be able to inhibit QS system of P. aeruginosa PAO1. Wogonin downregulated the expression of QS-related genes and reduced the production of many virulence factors, such as elastase, pyocyanin, and proteolytic enzyme. In addition, wogonin decreased the extracellular polysaccharide synthesis and inhibited twitching, swimming, and swarming motilities and biofilm formation. The attenuation of pathogenicity in P. aeruginosa PAO1 by wogonin application was further validated in vivo by cabbage infection and fruit fly and nematode survival experiments. Further molecular docking analysis, pathogenicity examination of various QS-related mutants, and PQS signal molecule detection revealed that wogonin could interfere with PQS signal molecular synthesis by affecting pqsA and pqsR. Taken together, the results indicated that wogonin might be used as an anti-QS candidate drug to attenuate the infection caused by P. aeruginosa.     

Molecular Characterization of Three Tandemly Located Flagellin Genes of Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is a motile, opportunistic pathogen. The flagellum, which is involved in swimming, swarming, adhesion, and biofilm formation, is considered a virulence factor for motile pathogens. Three flagellin genes, fliC1, fliC2, and fliC3, were identified from the sequenced S. maltophilia genome. FliC1, fliC2, and fliC3 formed an operon, and their encoding proteins shared 67–82% identity. Members of the fliC1C2C3 operon were deleted individually or in combination to generate single mutants, double mutants, and a triple mutant. The contributions of the three flagellins to swimming, swarming, flagellum morphology, adhesion, and biofilm formation were assessed. The single mutants generally had a compromise in swimming and no significant defects in swarming, adhesion on biotic surfaces, and biofilm formation on abiotic surfaces. The double mutants displayed obvious defects in swimming and adhesion on abiotic and biotic surfaces. The flagellin-null mutant lost swimming ability and was compromised in adhesion and biofilm formation. All tested mutants demonstrated substantial but different flagellar morphologies, supporting that flagellin composition affects filament morphology. Bacterial swimming motility was significantly compromised under an oxidative stress condition, irrespective of flagellin composition. Collectively, the utilization of these three flagellins for filament assembly equips S. maltophilia with flagella adapted to provide better ability in swimming, adhesion, and biofilm formation for its pathogenesis.     

Biofilm Matrix and Its Regulation in Pseudomonas aeruginosa

Biofilms are communities of microorganisms embedded in extracellular polymeric substances (EPS) matrix. Bacteria in biofilms demonstrate distinct features from their free-living planktonic counterparts, such as different physiology and high resistance to immune system and antibiotics that render biofilm a source of chronic and persistent infections. A deeper understanding of biofilms will ultimately provide insights into the development of alternative treatment for biofilm infections. The opportunistic pathogen Pseudomonas aeruginosa, a model bacterium for biofilm research, is notorious for its ability to cause chronic infections by its high level of drug resistance involving the formation of biofilms. In this review, we summarize recent advances in biofilm formation, focusing on the biofilm matrix and its regulation in P. aeruginosa, aiming to provide resources for the understanding and control of bacterial biofilms.     

The Xanthophyll Carotenoid Lutein Reduces the Invasive Potential of Pseudomonas aeruginosa and Increases Its Susceptibility to Tobramycin

Recently, the xanthophyll carotenoid lutein has been qualified as a potential quorum sensing (QS) and biofilm inhibitor against Pseudomonas aeruginosa. To address the potential of this xanthophyll compound as a relevant antivirulence agent, we investigated in depth its impact on the invasion capabilities and aggressiveness of P. aeruginosa PAO1, which rely on the bacterial ability to build and maintain protective barriers, use different types of motilities and release myriad virulence factors, leading to host cell and tissue damages. Our data, obtained on the PAO1 strain, indicate that all-trans lutein (Lut; 22 µM) disrupts biofilm formation and disorganizes established biofilm structure without affecting bacterial viability, while improving the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Furthermore, this xanthophyll affects PAO1 twitching and swarming motilities while reducing the production of the extracellular virulence factors pyocyanin, elastase and rhamnolipids as well as the expression of the QS-regulated lasB and rhlA genes without inhibiting the QS-independent aceA gene. Interestingly, the expression of the QS regulators rhlR/I and lasR/I is significantly reduced as well as that of the global virulence factor regulator vfr, which is suggested to be a major target of Lut. Finally, an oxidative metabolite of Lut, 3′-dehydrolutein, induces a similar inhibition phenotype. Taken together, lutein-type compounds represent potential agents to control the invasive ability and antibiotic resistance of P. aeruginosa.     

Sodium Dodecyl Sulfate (SDS)-Loaded Nanoporous Polymer as Anti-Biofilm Surface Coating Material

Biofilms cause extensive damage to industrial settings. Thus, it is important to improve the existing techniques and develop new strategies to prevent bacterial biofilm formation. In the present study, we have prepared nanoporous polymer films from a self-assembled 1,2-polybutadiene-b-polydimethylsiloxane (1,2-PB-b-PDMS) block copolymer via chemical cross-linking of the 1,2-PB block followed by quantitative removal of the PDMS block. Sodium dodecyl sulfate (SDS) was loaded into the nanoporous 1,2-PB from aqueous solution. The SDS-loaded nanoporous polymer films were shown to block bacterial attachment in short-term (3 h) and significantly reduce biofilm formation in long-term (1 week) by gram-negative bacterium Escherichia coli. Tuning the thickness or surface morphology of the nanoporous polymer films allowed to extent the anti-biofilm capability.     

The Effect of a Silver Nanoparticle Polysaccharide System on Streptococcal and Saliva-Derived Biofilms

In this work, we studied the antimicrobial properties of a nanocomposite system based on a lactose-substituted chitosan and silver nanoparticles: Chitlac-nAg. Twofold serial dilutions of the colloidal Chitlac-nAg solution were both tested on Streptococcus mitis, Streptococcus mutans, and Streptococcus oralis planktonic phase and biofilm growth mode as well as on saliva samples. The minimum inhibitory and bactericidal concentrations of Chitlac-nAg were evaluated together with its effect on sessile cell viability, as well as both on biofilm formation and on preformed biofilm. In respect to the planktonic bacteria, Chitlac-nAg showed an inhibitory/bactericidal effect against all streptococcal strains at 0.1% (v/v), except for S. mitis ATCC 6249 that was inhibited at one step less. On preformed biofilm, Chitlac-nAg at a value of 0.2%, was able to inhibit the bacterial growth on the supernatant phase as well as on the mature biofilm. For S. mitis ATCC 6249, the biofilm inhibitory concentration of Chitlac-nAg was 0.1%. At sub-inhibitory concentrations, the Streptococcal strains adhesion capability on a polystyrene surface showed a general reduction following a concentration-dependent-way; a similar effect was obtained for the metabolic biofilm activity. From these results, Chitlac-nAg seems to be a promising antibacterial and antibiofilm agent able to hinder plaque formation.     

Aspergillus parasiticus crzA,Which Encodes Calcineurin Response Zinc-Finger Protein,Is Required for Aflatoxin Production under Calcium Stress

Two morphologically different Aspergillus parasiticus strains, one producing aflatoxins, abundant conidia but few sclerotia (BN9) and the other producing O-methyl-sterimatocystin (OMST), copious sclerotia but a low number of conidia (RH), were used to assess the role of crzA which encodes a putative calcium-signaling pathway regulatory protein. Under standard culture conditions, BN9ΔcrzA mutants conidiated normally but decreased slightly in radial growth, regardless of illumination conditions. RHΔcrzA mutants produced only conidia under light and showed decreased conidiation and delayed sclerotial formation in the dark. Regulation of conidiation of both A. parasiticus strains by light was independent of crzA. Increased concentrations of lithium, sodium, and potassium impaired conidiation and sclerotial formation of the RHΔcrzA mutants but they did not affect conidiation of the BN9ΔcrzA mutants. Vegetative growth and asexual development of both ΔcrzA mutants were hypersensitive to increased calcium concentrations. Calcium supplementation (10 mM) resulted in 3-fold and 2-fold decreases in the relative expression of the endoplasmic reticulum calcium ATPase 2 gene in the BN9 and RH parental strains, respectively, but changes in both ΔcrzA mutants were less significant. Compared to the parental strains, the ΔcrzA mutants barely produced aflatoxins or OMST after the calcium supplementation. The relative expression levels of aflatoxin biosynthesis genes, nor1, ver1, and omtA, in both ΔcrzA mutants were decreased significantly, but the decreases in the parental strains were at much lower extents. CrzA is required for growth and development and for aflatoxin biosynthesis under calcium stress conditions.     

The Interaction of CuS and Halothiobacillus HT1 Biofilm in Microscale Using Synchrotron Radiation-Based Techniques

In order to investigate the microbe-mineral interaction in the micro scale, spatial distribution and speciation of Cu and S in Halothiobacillus HT1 biofilm formed on a CuS surface was examined using synchrotron-based X-ray techniques. Confocal laser scanning microscope (CLSM) results indicated that Halothiobacillus HT1 biofilm formation gave rise to distinct chemical and redox gradients, leading to diverse niches in the biofilm. Live cells were distributed at the air-biofilm and membrane-biofilm interface. CuS was oxidized by Halothiobacillus HT1 biofilm, and copper penetrated into the biofilm. Sulfide was oxidized to cysteine (77.3%), sulfite (3.8%) and sulfonate (18.9%). Cu-cysteine-like species were involved in the copper homeostasis. These results significantly improve our understanding of the interfacial properties of the biofilm-mineral interface.     

Role of Candida albicans-Secreted Aspartyl Proteinases (Saps) in Severe Early Childhood Caries

Candida albicans is strongly associated with severe early childhood caries (S-ECC). However, the roles of secreted aspartyl proteinases (Saps), an important virulence factor of C. albicans, in the progress of S-ECC are not clear. In our study, the Saps activities were evaluated by the yeast nitrogen base–bovine serum albumi (YNB–BSA) agar plate method and by the MTT method with bovine serum albumin (BSA) as the substrate. Genotypes of C. albicans and gene expression of Sap1–5 were evaluated. The relationships of Saps activities and genotypes with S-ECC were analyzed. The results showed that enzyme activities of Saps in the S-ECC group were significantly higher than those in the caries free (CF) group (p < 0.05). Genotypes A, B and C were detected in the S-ECC group, and genotypes A and C were detected in the CF group. In the genotype A group, Saps activity in the S-ECC group was significantly different from that in the CF group (p < 0.05). The gene expression level of Sap1 in the S-ECC group was significantly higher than that in the CF group (p = 0.001), while Sap4 expression was significantly lower than that in the CF group (p = 0.029). It can be concluded that Sap1–5 are the predominant proteinase genes expressed in C. albicans from dental biofilm and Sap1 may play an important role in the development of S-ECC.     

(+)-Dehydroabietic Acid,an Abietane-Type Diterpene,Inhibits Staphylococcus aureus Biofilms in Vitro

Potent drugs are desperately needed to counteract bacterial biofilm infections, especially those caused by gram-positive organisms, such as Staphylococcus aureus. Moreover, anti-biofilm compounds/agents that can be used as chemical tools are also needed for basic in vitro or in vivo studies aimed at exploring biofilms behavior and functionability. In this contribution, a collection of naturally-occurring abietane-type diterpenes and their derivatives was tested against S. aureus biofilms using a platform consisting of two phenotypic assays that have been previously published by our group. Three active compounds were identified: nordehydroabietylamine (1), (+)-dehydroabietic acid (2) and (+)-dehydroabietylamine (3) that prevented biofilm formation in the low micromolar range, and unlike typical antibiotics, only 2 to 4-fold higher concentrations were needed to significantly reduce viability and biomass of existing biofilms. Compound 2, (+)-dehydroabietic acid, was the most selective towards biofilm bacteria, achieving high killing efficacy (based on log Reduction values) and it was best tolerated by three different mammalian cell lines. Since (+)-dehydroabietic acid is an easily available compound, it holds great potential to be used as a molecular probe in biofilms-related studies as well as to serve as inspirational chemical model for the development of potent drug candidates.