Baolin Sun

Lipase-Sensitive Polymeric Triple-Layered Nanogel for “On-Demand” Drug Delivery

We report a new strategy for differential delivery of antimicrobials to bacterial infection sites with a lipase-sensitive polymeric triple-layered nanogel (TLN) as the drug carrier. The TLN was synthesized by a convenient arm-first procedure using an amphiphilic diblock copolymer, namely, monomethoxy poly(ethylene glycol)-b-poly(ε-caprolactone), to initiate the ring-opening polymerization of the difunctional monomer 3-oxapentane-1,5-diyl bis(ethylene phosphate). The hydrophobic poly(ε-caprolactone) (PCL) segments collapsed and surrounded the polyphosphoester core, forming a hydrophobic and compact molecular fence in aqueous solution which prevented antibiotic release from the polyphosphoester core prior to reaching bacterial infection sites. However, once the TLN sensed the lipase-secreting bacteria, the PCL fence of the TLN degraded to release the antibiotic. Using Staphylococcus aureus (S. aureus) as the model bacterium and vancomycin as the model antimicrobial, we demonstrated that the TLN released almost all the encapsulated vancomycin within 24 h only in the presence of S. aureus, significantly inhibiting S. aureus growth. The TLN further delivered the drug into bacteria-infected cells and efficiently released the drug to kill intracellular bacteria. This technique can be generalized to selectively deliver a variety of antibiotics for the treatment of various infections caused by lipase-secreting bacteria and thus provides a new, safe, effective, and universal approach for the treatment of extracellular and intracellular bacterial infections.

Isolation and Characterization of Desulfovibrio dechloracetivorans sp. nov., a Marine Dechlorinating Bacterium Growing by Coupling the Oxidation of Acetate to the Reductive Dechlorination of 2-Chlorophenol

ABSTRACT Strain SF3, a gram-negative, anaerobic, motile, short curved rod that grows by coupling the reductive dechlorination of 2-chlorophenol (2-CP) to the oxidation of acetate, was isolated from San Francisco Bay sediment. Strain SF3 grew at concentrations of NaCl ranging from 0.16 to 2.5%, but concentrations of KCl above 0.32% inhibited growth. The isolate used acetate, fumarate, lactate, propionate, pyruvate, alanine, and ethanol as electron donors for growth coupled to reductive dechlorination. Among the halogenated aromatic compounds tested, only the ortho position of chlorophenols was reductively dechlorinated, and additional chlorines at other positions blockedortho dechlorination. Sulfate, sulfite, thiosulfate, and nitrate were also used as electron acceptors for growth. The optimal temperature for growth was 30°C, and no growth or dechlorination activity was observed at 37°C. Growth by reductive dechlorination was revealed by a growth yield of about 1 g of protein per mol of 2-CP dechlorinated, and about 2.7 g of protein per mole of 2,6-dichlorophenol dechlorinated. The physiological features and 16S ribosomal DNA sequence suggest that the organism is a novel species of the genus Desulfovibrio and which we have designatedDesulfovibrio dechloracetivorans. The unusual physiological feature of this strain is that it uses acetate as an electron donor and carbon source for growth with 2-CP but not with sulfate.

Identification of two novel esterases from a marine metagenomic library derived from South China Sea

The demand for novel biocatalysts is increasing in modern biotechnology, which greatly stimulates the development of powerful tools to explore the genetic resources in the environment. Metagenomics, a culture independent strategy, provides an access to valuable genetic resources of the uncultured microbes. In this study, two novel esterase genes designated as estA and estB, which encoded 277- and 328-amino-acid peptides, respectively, were isolated from a marine microbial metagenomic library by functional screening, and the corresponding esterases EstA and EstB were biochemically characterized. Amino acid sequence comparison and phylogenetic analysis indicated that EstA together with other putative lipolytic enzymes was closely related to family III, and EstB with its relatives formed a subfamily of family IV. Site-directed mutagenesis showed that EstA contained classical catalytic triad made up of S146–D222–H255, whereas EstB contained an unusual catalytic triad which consisted of S–E–H, an important feature of the subfamily. EstA exhibited habitat-specific characteristics such as its high level of stability in the presence of various divalent cations and at high concentrations of NaCl. EstB displayed remarkable activity against p-nitrophenyl esters and was highly stable in 30% methanol, ethanol, dimethylformamide, and dimethyl sulfoxide, making EstB a potential candidate for industrial applications.

Staphylococcus aureus AI-2 Quorum Sensing Associates with the KdpDE Two-Component System To Regulate Capsular Polysaccharide Synthesis and Virulence

ABSTRACT Autoinducer 2 (AI-2) is widely recognized as a signal molecule for intra- and interspecies communication in Gram-negative bacteria, but its signaling function in Gram-positive bacteria, especially in Staphylococcus aureus, remains obscure. Here we reveal the role of LuxS in the regulation of capsular polysaccharide synthesis in S. aureus NCTC8325 and show that AI-2 can regulate gene expression and is involved in some physiological activities in S. aureus as a signaling molecule. Inactivation of luxS in S. aureus NCTC8325 resulted in higher levels of transcription of capsular polysaccharide synthesis genes. The survival rate of the luxS mutant was higher than that of the wild type in both human blood and U937 macrophages. In comparison to the luxS mutant, a culture supplemented with chemically synthesized 4,5-dihydroxy-2,3-pentanedione (DPD), the AI-2 precursor molecule, restored all the parental phenotypes, suggesting that AI-2 has a signaling function in S. aureus. Furthermore, we demonstrated that the LuxS/AI-2 signaling system regulates capsular polysaccharide production via a two-component system, KdpDE, whose function has not yet been clarified in S. aureus. This regulation occurred via the phosphorylation of KdpE binding to the cap promoter.

Staphylococcus aureus autoinducer-2 quorum sensing decreases biofilm formation in an icaR-dependent manner

BackgroundStaphylococcus aureus is an important pathogen that causes biofilm-associated infection in humans. Autoinducer 2 (AI-2), a quorum-sensing (QS) signal for interspecies communication, has a wide range of regulatory functions in both Gram-positive and Gram-negative bacteria, but its exact role in biofilm formation in S. aureus remains unclear.ResultsHere we demonstrate that mutation of the AI-2 synthase gene luxS in S. aureus RN6390B results in increased biofilm formation compared with the wild-type (WT) strain under static, flowing and anaerobic conditions and in a mouse model. Addition of the chemically synthesized AI-2 precursor in the luxS mutation strain (ΔluxS) restored the WT phenotype. Real-time RT-PCR analysis showed that AI-2 activated the transcription of icaR, a repressor of the ica operon, and subsequently a decreased level of icaA transcription, which was presumably the main reason why luxS mutation influences biofilm formation. Furthermore, we compared the roles of the agr-mediated QS system and the LuxS/AI-2 QS system in the regulation of biofilm formation using the ΔluxS strain, RN6911 and the Δagr ΔluxS strain. Our data indicate a cumulative effect of the two QS systems on the regulation of biofilm formation in S. aureus.ConclusionThese findings demonstrate that AI-2 can decrease biofilm formation in S. aureus via an icaR-activation pathway. This study may provide clues for therapy in S. aureus biofilm-associated infection.

The Staphylococcus aureus KdpDE Two-Component System Couples Extracellular K+ Sensing and Agr Signaling to Infection Programming

ABSTRACT The Kdp system is widely distributed among bacteria. In Escherichia coli, the Kdp-ATPase is a high-affinity K+ uptake system and its expression is activated by the KdpDE two-component system in response to K+ limitation or salt stress. However, information about the role of this system in many bacteria still remains obscure. Here we demonstrate that KdpFABC in Staphylococcus aureus is not a major K+ transporter and that the main function of KdpDE is not associated with K+ transport but that instead it regulates transcription for a series of virulence factors through sensing external K+ concentrations, indicating that this bacterium might modulate its infectious status through sensing specific external K+ stimuli in different environments. Our results further reveal that S. aureus KdpDE is upregulated by the Agr/RNAIII system, which suggests that KdpDE may be an important virulence regulator coordinating the external K+ sensing and Agr signaling during pathogenesis in this bacterium.

LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing

In quorum sensing (QS) process, bacteria regulate gene expression by utilizing small signaling molecules called autoinducers in response to a variety of environmental cues. Autoinducer 2 (AI-2), a QS signaling molecule proposed to be involved in interspecies communication, is produced by many species of gram-negative and gram-positive bacteria. In Escherichia coli and Salmonella typhimurium, the extracellular AI-2 is imported into the cell by a transporter encoded by the lsr operon. Upstream of the lsr operon, there is a divergently transcribed gene encoding LsrR, which was reported previously to repress the transcription of the lsr operon and itself. Here, we have demonstrated for the first time that LsrR represses the transcription of the lsr operon and itself by directly binding to their promoters using gel shift and DNase I footprinting assays. The β-galactosidase reporter assays further suggest that two motifs in both the lsrR and lsrA promoter regions are crucial for the LsrR binding. Furthermore, in agreement with the conclusion that phosphorylated AI-2 can relieve the repression of LsrR in previous studies, our data show that phospho-AI-2 renders LsrR unable to bind to its own promoter in vitro.

Desulfomonile limimaris sp. nov., an anaerobic dehalogenating bacterium from marine sediments

Strains DCB-MT and DCB-F were isolated from anaerobic 3-chlorobenzoate (3CB)-mineralizing cultures enriched from marine sediments. The isolates are large, Gram-negative rods with a collar girdling each cell. The isolates are obligate anaerobes capable of reductive dechlorination of 3CB to benzoate. Growth by chlororespiration in strain DCB-MT yielded 1.7 g protein mol(-1) 3CB dechlorinated with lactate as the electron donor. Strain DCB-MT also used fumarate, sulfate, sulfite, thiosulfate and nitrate as physiological electron acceptors for growth, but grew poorly on sulfate and nitrate. Reductive dechlorination was inhibited completely by sulfite and thiosulfate but not by sulfate. Both strains were incapable of growth at NaCl concentrations below 0.32% (w/v). They grew well at sea-water salt concentrations; however, the optimum growth rate was achieved at a NaCl concentration half that of sea water. The 16S rDNA sequence analysis shows strains DCB-MT and DCB-F to be 99% similar to each other and 93% similar to their closest relative, Desulfomonile tiedjei strain DCB-1T. Strain DCB-MT can also be distinguished from strain DCB-1T by its inability to use acetate for growth on 3CB and by its requirement for NaCl. The morphology, physiology and 16S rDNA sequences of DCB-MT and DCB-F suggest that these strains represent a new, marine-adapted species of the genus Desulfomonile, designated Desulfomonile limimaris sp. nov. The type strain is strain DCB-MT (= ATCC 700979T).

Shewanella xiamenensis sp. nov., isolated from coastal sea sediment.

A Gram-negative, motile, rod-shaped bacterium, strain S4(T), was isolated from coastal sediment collected off Xiamen, China. The physiological and biochemical features of strain S4(T), determined using the API 20NE, API ZYM and Biolog GN2 systems, were similar to those of members of the genus Shewanella. Phylogenetic analyses based on 16S rRNA and gyrB gene sequences placed strain S4(T) in the genus Shewanella, and it was most closely related to Shewanella oneidensis and related species. DNA-DNA hybridization demonstrated only 11.9-30.4 % relatedness between S4(T) and the type strains of related Shewanella species. On the basis of phylogenetic and phenotypic characteristics, strain S4(T) is classified in the genus Shewanella as a representative of a distinct novel species, for which the name Shewanella xiamenensis sp. nov. is proposed. The type strain is S4(T) (=CCTCC M 209017(T) =JCM 16212(T)).

The Staphylococcus aureus GGDEF Domain-Containing Protein, GdpS, Influences Protein A Gene Expression in a Cyclic Diguanylic Acid-Independent Manner

ABSTRACT Staphylococcus aureus is an important human pathogen that is the principal cause of a variety of diseases, ranging from localized skin infections to life-threatening systemic infections. The success of the organism as a pathogen and its ability to cause such a wide range of infections are due to its extensive virulence factors. In this study, we identified the role of the only GGDEF domain protein (GdpS [GGDEF domain protein from Staphylococcus]) in the virulence of S. aureus NCTC8325. Inactivation of gdpS results in an alteration in the production of a range of virulence factors, such as serine and cysteine proteases, fibrinogen-binding proteins, and, specifically, protein A (Spa), a major surface protein of S. aureus. The transcript level of spa decreases eightfold in the gdpS mutant compared with the parental NCTC8325 strain. Furthermore, the transcript level of sarS, which encodes a direct positive regulator of spa, also decreases in the gdpS mutant compared with the wild type, while the transcript levels of agr, sarA, sarT, and rot display no apparent changes in the gdpS mutant, suggesting that GdpS affects the expression of spa through interaction with SarS by unknown mechanisms. Furthermore, the complementation assays show that the influences of GdpS on spa and sarS depend on its N-terminal domain, which is predicted to be the sensor of a two-component system, rather than its C-terminal GGDEF domain with conserved GGDEF, suggesting that GdpS functions in S. aureus by an unknown mechanism independent of 3′,5′-cyclic diguanylic acid signaling.