Addition of L-cysteine to the N- or C-terminus of the all-D-enantiomer [D(KLAKLAK)2] increases antimicrobial activities against multidrug-resistant Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli.

BACKGROUND: Antimicrobial peptides have a broad spectrum of antimicrobial activities and are attracting attention as promising next-generation antibiotics against multidrug-resistant (MDR) bacteria. The all-d-enantiomer [D(KLAKLAK)2] has been reported to have antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa, and to be resistant to protein degradation in bacteria because it is composed of D-enantiomer compounds. In this study, we demonstrated that modification of [D(KLAKLAK)2] by the addition of an L-cysteine residue to its N- or C- terminus markedly enhanced its antimicrobial activities against Gram-negative bacteria such as MDR Acinetobacter baumannii, E. coli, and P. aeruginosa. METHODS: The peptides [D(KLAKLAK)2] (DP), DP to which L-cysteine was added at the N-terminus C-DP, and DP to which L-cysteine was added at the C-terminus DP-C, were synthesized at >95% purity. The minimum inhibitory concentrations of peptides and antibiotics were determined by the broth microdilution method. The synergistic effects of the peptides and the antibiotics against MDR P. aeruginosa were evaluated using the checkerboard dilution method. In order to assess how these peptides affect the survival of human cells, cell viability was determined using a Cell Counting Kit-8. RESULTS: C-DP and DP-C enhanced the antimicrobial activities of the peptide against MDR Gram-negative bacteria, including A. baumannii, E. coli, and P. aeruginosa. The antimicrobial activity of DP-C was greater than that of C-DP, with these peptides also having antimicrobial activity against drug-susceptible P. aeruginosa and drug-resistant P. aeruginosa overexpressing the efflux pump components. C-DP and DP-C also showed antimicrobial activity against colistin-resistant E. coli harboring mcr-1, which encodes a lipid A modifying enzyme. DP-C showed synergistic antimicrobial activity against MDR P. aeruginosa when combined with colistin. The LD50 of DP-C against a human cell line HepG2 was six times higher than the MIC of DP-C against MDR P. aeruginosa. The LD50 of DP-C was not altered by incubation with low-dose colistin. CONCLUSION: Attachment of an L-cysteine residue to the N- or C-terminus of [D(KLAKLAK)2] enhanced its antimicrobial activity against A. baumannii, E. coli, and P. aeruginosa. The combination of C-DP or DP-C and colistin had synergistic effects against MDR P. aeruginosa. In addition, DP-C and C-DP showed much stronger antimicrobial activity against MDR A. baumannii and E. coli than against P. aeruginosa.

A peptide based on homologous sequences of the ß-barrel assembly machinery component BamD potentiates antibiotic susceptibility of Pseudomonas aeruginosa.

OBJECTIVES: The ß-barrel assembly machinery (BAM) complex plays a critical role in outer membrane protein (OMP) biogenesis. The outer membrane (OM) of Pseudomonas aeruginosa is centrally involved in mechanisms of antibiotic resistance. This study aimed to identify effects of a synthetic peptide based on conserved sequences in the putative BamA-binding region of BamD, focusing on antibiotic susceptibility and OMP characteristics in P. aeruginosa. METHODS: We synthesized a peptide FIRL (Phe-Ile-Arg-Leu-CONH(2)) with a sequence related to that of the BamD protein. We assessed antibiotic susceptibility of P. aeruginosa PAO1 using the chequerboard method and a time-kill assay. Changes in OMPs and in OM permeability were examined using SDS-PAGE, western blot analysis and nitrocefin assays. The combined effects of the peptide and antibiotics were investigated using a mouse pneumonia model. RESULTS: Although the peptide alone exerted no antimicrobial effect, it reduced the MICs of colistin, levofloxacin, erythromycin, vancomycin and rifampicin for P. aeruginosa PAO1 by 4-fold or more. Time-kill tests revealed bacterial numbers were significantly reduced after 2 h of incubation with the peptide plus colistin or levofloxacin. Moreover, in the presence of the peptide, expression of OprM was reduced by a third, and OM permeability was increased. The combination of the peptide (2.08 mg/kg) and colistin (1.25 mg/kg) significantly reduced P. aeruginosa by more than 1 log cfu/mL in a mouse pneumonia model. CONCLUSIONS: We show, for the first time, that a synthetic peptide based on homologous sequences of BamD can potentiate antibiotic susceptibility of P. aeruginosa.

Functional interaction sites of OprM with MexAB in the Pseudomonas aeruginosa multidrug efflux pump.

Subunit-swapping between Pseudomonas aeruginosa MexAB-OprM and MexEF-OprN efflux pumps has shown that OprM can interact with MexEF to produce a functional efflux pump, but that OprN cannot functionally interact with MexAB. Taking advantage of this subunit selectivity, we carried out experiments using chimeric proteins composed of OprM and OprN to determine which regions of OprM are necessary for functional interaction with MexAB. We constructed two types of chimeric proteins: one with the N-terminal half of OprM and the C-terminal half of OprN (OprMN), and the second with these halves reversed (OprNM). Introduction of either of the chimeric protein genes into a mutant expressing MexEF alone restored the functionality of the efflux pump. However, expression of OprMN or OprNM in the presence of MexAB did not restore the pump functionality, indicating that the both the N- and C-terminal halves of OprM are necessary for a functional interaction with MexAB.

Modification of a human monoclonal antibody Fab fragment specific for Plasmodium falciparum 19-kDa C-terminal merozoite surface protein 1 by site-directed mutagenesis.

We recently produced human monoclonal antibody Fab fragments specific for the 19-kDa C-terminal merozoite surface protein 1 of Plasmodium falciparum in a bacterial expression system. The effect of single amino acid modifications in the third complementarity-determining regions of the heavy and light chains on affinity was examined in one of the Fab fragments, Pf25. Recombination polymerase chain reaction was used to modify Tyr(92) or Ile(97) in the light chain and Val(101) or Trp(107) in the heavy chain. No effective replacements for Tyr(92) and Val(101) were found, but possible substitutions of Ile(97) with Gly, Leu, Glu, Ala and Ser, and of Trp(107) with Arg and Ser were demonstrated. Of these modified Fab fragments, the affinities of Fabs with Ile(97)-Leu and Trp(107)-Ser mutations were slightly higher than that of the original Fab. The effects of these modifications on the antigen-antibody interaction are discussed.

Diversity in the oligomeric channel structure of the multidrug efflux pumps in Pseudomonas aeruginosa.

MexAB-OprM, the multidrug efflux pump of Pseudomonas aeruginosa, contributes to the high resistance of this organism to a wide variety of antibiotics. To investigate the structure and function of OprM, the outer membrane channel of MexAB-OprM, we examined the oligomeric states of OprM and its homologues OprJ and OprN. These proteins were treated with crosslinking reagent after their reconstitution into liposome membranes. The crosslinked products indicated that OprM and OprN formed trimers, while OprJ unexpectedly appeared to form a tetramer. In order to test whether differences in oligomeric structure might be intimately related to channel function, we examined the channel-forming activity of these proteins by liposome swelling assay. However, no significant differences in channel characteristics were detected among OprM, OprJ, and OprN. We proposed the probable explanation for the diversity in the oligomeric structure of the channel proteins.

Assignment of the outer-membrane-subunit-selective domain of the membrane fusion protein in the tripartite xenobiotic efflux pump of Pseudomonas aeruginosa.

Early in vivo experiments revealed that the MexA-MexB dipartite pump unit of Pseudomonas aeruginosa conferred drug resistance to the cells, which expressed OprM, but not to the OprN-bearing cells. While the MexE-MexF unit interplayed with either the outer membrane subunits. Taking advantage of this subunit selectivity, we selected the MexA mutant that gained the ability to interplay with OprN. Four mutants have been isolated and all showed an amino acid substitution (Q116R) in the coiled-coil domain of MexA. The hybrid protein bearing the coiled-coil domain of MexA and the remainder domains from MexE retained the ability to interplay with OprM, but lost the functional interplay with OprN. These results established that the coiled-coil domain of the membrane fusion protein is responsible for selecting the compatible outer membrane subunit.

Multidrug transporter MexB of Pseudomonas aeruginosa: overexpression, purification, and initial structural characterization.

Structural and functional characterization of the multidrug transporter, MexB, of Pseudomonas aeruginosa is significantly restricted due to a low yield of approximately 0.1 mg/L of culture from natural sources. To facilitate structural studies of this medically important transporter protein, we developed a large-scale system for expression of the genetically engineered recombinant, MexB, in the Escherichia coli cell. Using the system, the eventual yield of MexB attained was about 10mg/L of culture. The optimized purification protocol in the presence of dodecyl beta-D-maltoside allowed isolation of highly homogeneous MexB. The oligomeric state of the protein in detergent solution has been characterized to verify that the native state of the purified protein has been preserved. The molecular mass of the protein-detergent complex was found to be 380-450kDa. The MexB-dodecyl beta-d-maltoside mass ratio was determined to be 1.8 +/- 0.05. Taking into account the monomeric MexB molecular mass deduced from its amino acid sequence (112.8 kDa), we concluded that the purified MexB exists as the homotrimer in the surfactant solution. Circular dichroism analysis of MexB showed dominance of the alpha-helix structures. High yield, homogeneity, and stability of MexB position it as a good candidate for structural and functional characterization.

Molecular characterization of peroxiredoxin from Entamoeba moshkovskii and a comparison with Entamoeba histolytica.

Peroxiredoxin of the pathogenic parasite, Entamoeba histolytica, is thought to be involved in protection from oxidative attack by host phagocytic cells and endogenously generated hydrogen peroxide. In this study, we cloned peroxiredoxin genes from the nonpathogenic ameba, Entamoeba moshkovskii, and characterized the peroxiredoxin protein. The open reading frame of three cloned cDNAs was demonstrated to encode a polypeptide of 218 or 217 amino acids. Identity of the amino acid sequence of peroxiredoxins between E. moshkovskii and E. histolytica was considerably high (77-81%), but the N-terminus portion of E. moshkovskii peroxiredoxin was shorter than that of E. histolytica. A recombinant peroxiredoxin of E. moshkovskii expressed in Escherichia coli exhibited hydrogen peroxidase activity. Its K(m) and V(max) values of 35 microM and 0.07 micromol/min/mg protein were approximately 1 and 1.5 times greater than E. histolytica peroxiredoxin, respectively. In addition, the protective effect of E. moshkovskii peroxiredoxin against oxidative-nicking of supercoiled plasmid DNA was shown to be greater than that of E. histolytica peroxiredoxin. Confocal laser scanning microscopy, using polyclonal antibody against the recombinant E. moshkovskii peroxiredoxin, demonstrated that this protein was localized in the nucleus and cytoplasm of trophozoites, supporting its function as a protectant against DNA damage. Southern blot and real-time reverse transcription PCR analyses of the E. moshkovskii peroxiredoxin gene demonstrated that it was a multi-copy gene and its expression was comparable to that of E. histolytica. These results suggest that the antioxidant peroxiredoxin is important for protection against endogenously generated hydrogen peroxide in the nonpathogenic ameba.

Improved affinity of a human anti-Entamoeba histolytica Gal/GalNAc lectin Fab fragment by a single amino acid modification of the light chain.

We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose- and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica. To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser91 and Arg96 in the third complementarity-determining region of the light chain with other amino acids. The screening of 200 clones of each exchange by an indirect fluorescent antibody test showed that 14 clones for Ser91 and nine clones for Arg96 reacted strongly with E. histolytica trophozoites. Sequence analyses revealed that the substituted amino acids at Ser91 were Ala in five clones, Gly in three clones, Pro in two clones, and Val in two clones, while the amino acid at position 96 was substituted with Leu in three clones. The remaining eight clones exhibited no amino acid change at position 91 or 96. These mutant Fab fragments were purified and subjected to a surface plasmon resonance assay to measure the affinity of these proteins to the cysteine-rich domain of lectin. Pro or Gly substitution for Ser91 caused an increased affinity of the Fab, but substitution with Ala or Val did not. The replacement of Arg96 with Leu did not affect affinity. These results demonstrate that modification of antibody genes by recombination PCR is a useful method for affinity maturation and that amino acid substitution at position 91 yields Fabs with increased affinity for the lectin.

MexZ-mediated regulation of mexXY multidrug efflux pump expression in Pseudomonas aeruginosa by binding on the mexZ-mexX intergenic DNA.

MexZ is a transcriptional regulator of the mexXY multidrug transporter operon, which confers aminoglycoside resistance on Pseudomonas aeruginosa. Highly purified MexZ showed direct binding with a specific site of the mexZ-mexX intergenic DNA when probed by a gel retardation assay. Both in vitro chemical cross-linking experiments and an in vivo two-hybrid expression system showed that the active form of MexZ, which is capable of binding the intergenic DNA, appeared to be a dimer. These results explain the mechanism by which MexZ represses transcription of the mexXY operon, but do not explain the substrate-induced hyperproduction of MexXY. The presence of inducer antibiotic in the gel-retardation assay mixture failed to detect altered MexZ-probe DNA interaction suggesting the possible involvement of an additional regulator.

Mutations affecting DNA-binding activity of the MexR repressor of mexR-mexA-mexB-oprM operon expression.

We have isolated 25 MexR mutants that retained their dimerizing ability but were unable to bind mexOP DNA. Surprisingly, 20 mutations were located in the hydrophobic core region at alpha4, W1, alpha2, alpha3, and beta2, and only 3 were in positively charged residues. These results verified that DNA binding is mediated by distinct regions of MexR and showed the importance of the hydrophobic core region of the DNA-binding domain.

Linkage of the efflux-pump expression level with substrate extrusion rate in the MexAB-OprM efflux pump of Pseudomonas aeruginosa.

The amount of the subunit proteins of the MexAB-OprM efflux pump in Pseudomonas aeruginosa was quantified by the immunoblotting method. A single cell of the wild-type strain contained about 2500, 1000, and 1200 copies of MexA, MexB, and OprM, respectively, and their stoichiometry therefore was 2:1:1. The mexR mutant produced an eightfold higher level of these proteins than did wild-type cells. Assuming that MexB and OprM exist as a trimer in a pump assembly, the total number of MexAB-OprM per wild-type cell was calculated to be about 400 assemblies. The substrate efflux rate of MexAB-OprM was calculated from the fluorescent intensity of ethidium in intact cells that a single cell extruded ethidium at a maximum of about 3 x 10(-19) mol s(-1) and, therefore, the turnover rate of a single pump unit was predicted to be about 500 s(-1).

A novel assembly process of the multicomponent xenobiotic efflux pump in Pseudomonas aeruginosa.

The nfxC-type cells of Pseudomonas aeruginosa show resistance to a wide range of structurally and functionally diverse antibiotics, which is a phenomenon that is mainly attributable to the expression of the MexEF-OprN xenobiotic transporter. The MexF, MexE and OprN subunits of this transporter are located on the inner membrane, the periplasm and the outer membrane, respectively, and are assumed to function as an energy-dependent transporter, a bridge connecting the inner and outer membranes and outer membrane channel respectively. The nfxC-type cells showed a single protein band of MexF and OprN, whereas MexE appeared as three distinct bands in an SDS-polyacrylamide gel electrophoretogram. The mutant cells lacking MexF produced undetectable OprN and only a full-size of MexE even though the cells had unimpaired oprN and mexE. Expression of the plasmid-borne MexF in this mutant fully restored OprN and three MexE bands. Another class of mutants producing a full amount of MexF yielded undetectable OprN and two MexE bands lacking the smallest protein species suggesting that the presence of the smallest MexE subunit is required for stabilization of OprN. To identify which part of MexE was needed for stabilization and assembly of OprN, the carboxyl-terminal-truncated MexE tagged with polyhistidine was constructed and protein bands were visualized in the presence of MexF with an antibody raised against polyhistidine or MexE. The results revealed that the proteolytic processing of MexE would occur at carboxyl terminal amino acids between 11 and 16, thereby suggesting that the presence of the C-terminal truncated MexE is essential for stabilization and the proper assembly of OprN. Nucleotide sequencing of mutant mexFs, which produce a wild-type level of MexF but are unable to support the production of the smallest MexE, thereby destabilizing OprN, revealed that all the mutations were located within two large periplasmic domains of MexF between transmembrane segments 1-2 and 7-8. Taking these findings together, we concluded that two large periplasmic domains of MexF interact with MexE thereby promoting programmed processing of MexE, and this complex eventually assists the correct assembly and sorting of OprN.

The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel.

OprM, the outer membrane component of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa, has been assumed to facilitate the export of antibiotics across the outer membrane of this organism. Here we purified to homogeneity the OprM protein, reconstituted it into liposome membranes, and tested its channel activity by using the liposome swelling assay. It was demonstrated that OprM is a channel-forming protein and exhibits the channel property that amino acids diffuse more efficiently than saccharides. However, antibiotics showed no significant diffusion through the OprM channel in the liposome membrane, suggesting that OprM functions as a gated channel. We reasoned that the protease treatment may cause the disturbance of the gate structure of OprM. Hence, we treated OprM reconstituted in the membranes with alpha-chymotrypsin and examined its solute permeability. The results demonstrated that the protease treatment caused the opening of an OprM channel through which antibiotics were able to diffuse. To elucidate which cleavage is intimately related to the opening, we constructed mutant OprM proteins where the amino acid at the cleavage site was replaced with another amino acid. By examining the channel activity of these mutant proteins, it was shown that the proteolysis at tyrosine 185 and tyrosine 196 of OprM caused the channel opening. Furthermore, these residues were shown to face into the periplasmic space and interact with other component(s). We considered the possible opening mechanism of the OprM channel based on the structure of TolC, a homologue of OprM.

Multiantibiotic resistance caused by active drug extrusion in hospital pathogens.

All living organisms from bacteria to mammals extrude noxious compounds to the external medium. When exposed to antibiotics, bacteria actively extrude intracellular antibiotic and develop resistance to the drug. Nosocomial Staphylococcus aureaus, Pseudomonas aeruginosa and other bacteria are resistant to a broad range of antibiotics and to structurally and functionally diverse chemotherapeutic agents and disinfectants. For this reason nosocomial infections are especially hard to treat in immunocompromised patients who may be infected by low-virulence bacteria. Extrusion-related antibiotic resistance in P. aeruginosa arises by the expression of Mex-extrusion pumps, including genetically distinct mexA-mexB-oprM, mexC-mexD-oprj, and mexE-mexF-oprN systems, each encoding two inner membrane proteins and one outer membrane protein. S. aureus becomes resistant to fluoroquinolone by expressing NorA extrusion proteins and to disinfectants by expressing Qac extrusion proteins. The drug extrusion machinery may be classified into several categories according to the number of transmembrane segments it exhibits. The proteins that belong to a major facilitator super family have 12 or 14 transmembrane segments. The extrusion proteins with molecular weight of 12,000 to 15,000 span the membrane 4 times and are collectively called small multidrug resistance proteins. The extrusion proteins that transport substrate across the inner and outer membrane of gram-negative bacteria are in the resistance nodulation cell division family.