Aya Kubo

Structural functions of antimicrobial long-chain alcohols and phenols☆

Antimicrobial activity of a series of long-chain alcohols and common naturally occurring alcohols were tested against 15 selected microorganisms in order to gain new insights into their structural functions. The maximum activity seems to depend on the hydrophobic chain length from the hydrophilic hydroxyl group, and also the microorganisms being tested. The results obtained with the alcohols exhibit a generally applicable rule to many other compounds.

Heparin-binding outer membrane protein of chlamydiae

As an intracellular pathogen, the mechanism by which Chlamydia invade eukaryotic cells represents a cornerstone to understanding chlamydial biology. The ability of chlamydiae specifically to bind heparan sulphate or heparin and the association of this ability to bind and enter mammalian host cells was approached by searching experimentally for chlamydial outer membrane proteins that bind heparin. The 60 000 molecular weight cysteine‐rich outer membrane complex protein, OmcB, bound heparin. The ability of OmcB to bind heparin was supported by mapping the region of the protein with heparin‐binding capacity and demonstrating that an OmcB synthetic 20‐mer peptide from this region specifically bound heparin. Surface localization of OmcB was shown using monospecific antisera specific to the 20‐mer OmcB peptide that bound the surfaces of elementary bodies (EB) and by heparin‐binding peptide cross‐linking of EB surface proteins.

Characterization and functional analysis of PorB, a Chlamydia porin and neutralizing target.

A predicted protein (CT713) with weak sequence similarity to the major outer membrane protein (20.4% identity) in Chlamydia trachomatis was identified by Chlamydia genome analysis. We show that this protein is expressed, surface accessible, localized to the chlamydial outer membrane complex and functions as a porin. This protein, PorB, was highly conserved among different serovars, with nearly identical sequences between serovars D, B, C and L2. Sequence comparison between C. trachomatis and Chlamydia pneumoniae showed less conservation between species with 59.3% identity. Immunofluorescence staining with monospecific antisera to purified PorB revealed antigen localized within chlamydial inclusions and found throughout the developmental cycle. Antibodies to PorB neutralized infectivity of C. trachomatis in an in vitro neutralization assay confirming that PorB is surface exposed. As PorB was found to be in the outer membrane, as well as having weak structural characteristics similar to major outer membrane protein (MOMP) and other porins, a liposome‐swelling assay was used to determine whether this protein had pore‐forming capabilities. PorB had pore‐forming activity and was shown to be different from MOMP porin activity.

Antibacterial activity of long-chain alcohols: the role of hydrophobic alkyl groups

The role of hydrophobic alkyl groups of long-chain alcohols to antibacterial activity against Staphylococcus aureus and Propionibacterium acnes is discussed. the maximum activity was found to deped on the chain length from the hydrophilic hydroxyl group, and also the bacteria tested.

Modes of antifungal action of alkanols against Saccharomyces cerevisiae

Primary aliphatic alcohols from C(6) to C(13) were tested for their antifungal activity against Saccharomyces cerevisiae. Undecanol was found to be the most potent fungicide followed by decanol. The time-kill curve study showed that undecanol was fungicidal against S. cerevisiae at any growth stages. This fungicidal activity was not influenced by pH values. The alcohols tested inhibited glucose-induced acidification by inhibiting the plasma membrane H(+)-ATPase. The primary antifungal action of amphipathic medium-chain (C(9)-C(12)) alkanols comes mainly from their ability as nonionic surfactants to disrupt the native membrane-associated function of the integral proteins. Hence, the antifungal activity of alkanols is mediated by biophysical process, and the maximum activity can be obtained when balance between hydrophilic and hydrophobic portions becomes the most appropriate.

Substrate-specific diffusion of select dicarboxylates through Chlamydia trachomatis PorB.

Chlamydiae contain two porins, MOMP and PorB, that facilitate diffusion of solutes through the outer membrane. MOMP is a general porin that permits the diffusion of a wide variety of compounds including carbohydrates and amino acids. The relative inefficiency of PorB as a general porin and its low abundance in the outer membrane suggest that it may function as a substrate-specific porin. The tricarboxylic acid (TCA) cycle of chlamydiae is incomplete and to function would require the exogenous acquisition of 2-oxoglutarate or glutamate. A liposome-swelling assay for anions as well as an enzyme-linked liposome assay were used to demonstrate the efficient diffusion of dicarboxylates such as 2-oxoglutarate through PorB. These data demonstrate that PorB is a dicarboxylate-specific porin that may feed the chlamydial TCA cycle and provide chlamydiae with carbon and energy production intermediates.

Design, expression and functional characterization of a synthetic gene encoding the Chlamydia trachomatis major outer membrane protein.

A synthetic gene coding for the Chlamydia trachomatis serovar L2 major outer membrane protein (MOMP) was designed, constructed and expressed in Escherichia coli. The native amino acid sequence was reverse translated and the resulting nucleotide combinations manipulated in order to evenly distribute 25 unique restriction sites along the length of the gene while retaining the native amino acid sequence. The synthetic gene was cloned into a T7 promoter-controlled plasmid (pET-3a) and the expressed product was analyzed to assess antigenicity, cellular localization and function. Monoclonal antibodies specific for native MOMP reacted to the expressed product by immunoblot. Outer membrane fractionation confirmed that the processed protein was located in the outer membrane. MOMP expressed in E. coli and present in the outer membrane was shown to function as a general diffusion porin. This system provides the means to produce readily modifiable MOMP either in purified form or as a membrane-associated protein, and so facilitate the investigation of its functional, structural and antigenic properties.

Anti-Salmonella activity of (2E)-alkenals

Aims:  This study investigated the effect of a series of naturally occurring aliphatic (2E)‐alkenals against Salmonella choleraesuis subsp. choleraesuis ATCC 35640 and evaluated their antibacterial action.