Biomedical subjects

W M Shafer

Publications and source records attributed to W M Shafer.

At least 19 recordsLinked to original sources

PubMed2004

Porphyrin-based compounds exert antibacterial action against the sexually transmitted pathogens Neisseria gonorrhoeae and Haemophilus ducreyi.

A series of porphyrin based compounds without (nMP) or with (MP) metals were found to have potent bactericidal action in vitro against the sexually transmitted pathogens Neisseria gonorrhoeae and Haemophilus ducreyi. nMP and MP did not show bactericidal activity against five species of lactobacilli. An MP containing gallium had the capacity to block a gonococcal infection in a murine vaginal model, indicating that its development as a topical microbicide to block sexually transmitted bacterial infections is warranted. In contrast to other bacterial species, loss of the gonococcal haemoglobin uptake system encoded by hpuB or energy supplied through the TonB-ExbB-ExbD system did not significantly affect levels of MP-susceptibility in gonococci. In contrast, mutations in gonococci that inactivate the mtrCDE-encoded efflux pump were found to enhance gonococcal susceptibility to nMPs and MPs while over-production of this efflux pump decreased levels of gonococcal susceptibility to these compounds.

Animals↗

PubMed2003

FarR regulates the farAB-encoded efflux pump of Neisseria gonorrhoeae via an MtrR regulatory mechanism.

The farAB operon of Neisseria gonorrhoeae encodes an efflux pump which mediates gonococcal resistance to antibacterial fatty acids. It was previously observed that expression of the farAB operon was positively regulated by MtrR, which is a repressor of the mtrCDE-encoded efflux pump system (E.-H. Lee and W. M. Shafer, Mol. Microbiol. 33:839-845, 1999). This regulation was believed to be indirect since MtrR did not bind to the farAB promoter. In this study, computer analysis of the gonococcal genome sequence database, lacZ reporter fusions, and gel mobility shift assays were used to elucidate the regulatory mechanism by which expression of the farAB operon is modulated by MtrR in gonococci. We identified a regulatory protein belonging to the MarR family of transcriptional repressors and found that it negatively controls expression of farAB by directly binding to the farAB promoter. We designated this regulator FarR to signify its role in regulating the farAB operon. We found that MtrR binds to the farR promoter, thereby repressing farR expression. Hence, MtrR regulates farAB in a positive fashion by modulating farR expression. This MtrR regulatory cascade seems to play an important role in adjusting levels of the FarAB and MtrCDE efflux pumps to prevent their excess expression in gonococci.

Amino Acid Transport Systems, Neutral↗

PubMed2002

Tailoring an antibacterial peptide of human lysosomal cathepsin G to enhance its broad-spectrum action against antibiotic-resistant bacterial pathogens.

Neutrophils contain several cationic antimicrobial proteins or peptides (CAPs) that exert antibiotic-like action against bacteria. These host-derived antibiotics kill susceptible bacteria by oxygen-independent mechanisms. Considerable interest in their activity has been generated in recent years due not only to their likely important role in innate host defense against infection, but also their possible use as therapeutic agents in treating infections caused by antibiotic-resistant pathogens. We have studied the antibacterial properties of human lysosomal cathepsin G (cat G). This highly cationic serine protease contains at least three antibacterial regions that by themselves can exert antibacterial action against Gram-negative bacteria, such as Pseudomonas aeruginosa. Only one of these peptides, defined by residues 117-136 of full-length cat G, has bactericidal action against Gram-positive pathogens, such as Staphylococcus aureus. Due to the broad-spectrum antibacterial action of this peptide, we have sought to define the amino acids within its primary sequence required for this activity and have developed variants with improved activity. This review emphasizes the importance of both cationicity and hydrophobicity as necessary characteristics for the antibacterial action of CAPs. It also proposes the strategy that naturally occurring large human CAPs can be dissected to smaller CAPs and then modified to enhance their activity in vitro. This approach could prove beneficial to those interested in developing antimicrobial peptides as therapeutic agents.

Amino Acid Sequence↗

PubMed2001

Decreased susceptibility to azithromycin and erythromycin mediated by a novel mtr(R) promoter mutation in Neisseria gonorrhoeae.

During a screen of Neisseria gonorrhoeae clinical isolates obtained in Uruguay for susceptibility to azithromycin, we noticed that approximately 10% of the strains examined displayed decreased susceptibility to azithromycin and erythromycin due to the mtr(CDE)-encoded efflux pump system, but remained susceptible to Triton X-100. We now report that the mtr(R) promoter region of one of these isolates contains a dinucleotide insertion (TT) that mediates this resistance phenotype.

Anti-Bacterial Agents↗

PubMed2001

A putatively phase variable gene (dca) required for natural competence in Neisseria gonorrhoeae but not Neisseria meningitidis is located within the division cell wall (dcw) gene cluster.

A cluster of 18 open reading frames (ORFs), 15 of which are homologous to genes involved in division and cell wall synthesis, has been identified in Neisseria gonorrhoeae and Neisseria meningitidis. The three additional ORFs, internal to the dcw cluster, are not homologous to dcw-related genes present in other bacterial species. Analysis of the N. meningitidis strain MC58 genome for foreign DNA suggests that these additional ORFs have not been acquired by recent horizontal exchange, indicating that they are a long-standing, integral part of the neisserial dcw gene cluster. Reverse transcription-PCR analysis of RNA extracted from N. gonorrhoeae strain FA19 confirmed that all three ORFs are transcribed in gonococci. One of these ORFs (dca, for division cluster competence associated), located between murE and murF, was studied in detail and found to be essential for competence in the gonococcal but not in the meningococcal strains tested. Computer analysis predicts that dca encodes an inner membrane protein similar to hypothetical proteins produced by other gram-negative bacteria. In some meningococcal strains dca is prematurely terminated following a homopolymeric tract of G's, the length of which differs between isolates of N. meningitidis, suggesting that dca is phase variable in this species. A deletion and insertional mutation was made in the dca gene of N. gonorrhoeae strain FA19 and N. meningitidis strain NMB. This mutation abrogated the ability of the gonococci to be transformed with chromosomal DNA. Thus, we conclude that the dca-encoded gene product is an essential competence factor for gonococci.

Amino Acid Sequence↗

PubMed2001

Genetic organization and regulation of antimicrobial efflux systems possessed by Neisseria gonorrhoeae and Neisseria meningitidis.

Efflux pumps can make a significant contribution to the capacity of bacteria to resist the action of antibiotics. Certain efflux pumps also recognize antimicrobial agents that are present in their respective hosts and their ability to export toxic agents could enhance bacterial survival during infection prior to appearance of cellular or humoral host defensive systems. This review is concerned with the principal efflux pumps possessed by two closely related strict human pathogens, Neisseria gonorrhoeae and Neisseria meningitidis. Specific emphasis is placed on the organization of the structural genes encoding the mtr and far efflux pumps, the substrates (often host-derived) recognized by these pumps, and the cis- and trans-acting transcriptional factors that regulate efflux pump gene expression in gonococci and meningococci. The overriding theme of this review is that the efflux pumps possessed by these pathogens likely contribute to their pathogenic mechanisms by providing a means to escape a number of antimicrobial compounds that bathe mucosal surfaces.

Anti-Bacterial Agents↗

PubMed2000

Gonorrhea among men who have sex with men: outbreak caused by a single genotype of erythromycin-resistant Neisseria gonorrhoeae with a single-base pair deletion in the mtrR promoter region.

During 1995-1997, an outbreak of 66 cases of gonorrhea caused by an erythromycin-resistant (Ery(r); MIC >/=1.0 microgram/mL) prototrophic (proto) auxotype IB-1 serovar of Neisseria gonorrhoeae occurred in King County, Washington; 65 cases involved men who have sex with men (MSM), which accounted for approximately 37% of infections among MSM during this period. Isolates from 19 of these 65 cases of infection were analyzed by DNA sequencing of the polymerase chain reaction-amplified promoter region of the mtrR gene and by pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA after NheI and SpeI digestion. Eighteen of the 19 isolates had a 1-bp A/T deletion in a 13-bp inverted repeat of the mtrR promoter region and shared a single PFGE type. Among MSM who provided data about sexual behavior, 37 (64%) of 58 MSM infected by the proto/IB-1 Ery(r) strain reported having >2 sex partners during the past 60 days, compared with 32 (30%) of 106 MSM infected by other strains (P<.001). This clonal outbreak of gonorrhea illustrates the ongoing need for behavioral preventive interventions among MSM.

Bacterial Proteins↗

PubMed1999

Induction of the mtrCDE-encoded efflux pump system of Neisseria gonorrhoeae requires MtrA, an AraC-like protein.

The mtr (multiple transferable resistance) gene complex in Neisseria gonorrhoeae encodes an energy-dependent efflux pump composed of the MtrC-MtrD-MtrE cell envelope proteins that serves to export structurally diverse antimicrobial, hydrophobic agents (HAs). Many of these agents have membrane-acting detergent activity. Using Triton X-100 (TX-100) as a representative HA, we found that the mtrCDE efflux pump operon could be induced to higher levels of expression when an HA-sensitive strain was exposed to sublethal concentrations of this non-ionic detergent and the structurally related spermicide, nonoxynol-9. This induction was at the level of mtrCDE gene transcription and was independent of the MtrR repressor, which normally decreases mtrCDE gene expression. However, the enhanced resistance of gonococci to TX-100 was dependent on the expression of a previously undescribed gonococcal protein that belonged to the AraC/XylS family of transcriptional activators. We have termed this protein MtrA to signify its likely role in the activation of mtrCDE gene expression. Taken together with previous studies dealing with the genetic control of mtrCDE gene expression, we propose that gonococci can modulate their resistance to HAs through both positive and negative transcriptional control processes. The action of these regulatory processes is probably of importance in determining the survival capacity of gonococci at mucosal surfaces that contain detergent-like HAs.

ATP-Binding Cassette Transporters↗

PubMed1999

The farAB-encoded efflux pump mediates resistance of gonococci to long-chained antibacterial fatty acids.

Gonococci often infect mucosal surfaces bathed in antibacterial fatty acids (FAs). Resistance of gonococci to FAs and other antibacterial hydrophobic agents has been attributed to the mtrCDE-encoded efflux pump system and a heretofore undefined mechanism. This alternative resistance mechanism has been suggested to mediate gonococcal resistance to long-chained FAs independently of the mtr efflux pump. We have now identified this alternative FA resistance system in gonococci and report that it bears significant similarity to the emrAB-encoded efflux pump possessed by Escherichia coli and the vceAB-encoded pump of Vibrio cholerae. We termed the gonococcal version of this efflux pump farAB (fatty acid resistance) to signify its involvement in FA resistance expressed by gonococci and to distinguish it from the emrAB- or vceAB-encoded pumps that modulate bacterial susceptibility to uncoupling agents and certain antibiotics. Although the farAB system in gonococci was found to provide resistance to FAs independently of the mtrCDE-encoded efflux pump, its function was dependent on the MtrE outer membrane protein. Moreover, expression of the tandemly linked farA and farB genes was positively associated with the presence of the MtrR transcriptional regulatory protein that normally downregulates the expression of mtrCDE. Thus, the data presented herein suggest that, while the mtrCDE- and farAB-encoded systems act independently to mediate resistance of gonococci to host-derived, hydrophobic antimicrobial agents, their capacity to export these agents is dependent on the same outer membrane protein (MtrE), and their expression may be differentially controlled by the same transcriptional regulatory protein (MtrR).

Amino Acid Transport Systems, Neutral↗

PubMed1999

Decreased azithromycin susceptibility of Neisseria gonorrhoeae due to mtrR mutations.

Single-dose azithromycin therapy has recently been used in Uruguay for the treatment of uncomplicated gonococcal infections. As part of an active surveillance study to monitor the emergence of antibiotic resistance in gonococcal isolates, we examined the levels of azithromycin susceptibility in 51 consecutive isolates obtained from males with uncomplicated gonococcal urethritis. Isolates with decreased susceptibility to azithromycin (MICs, 0.25 to 0.5 microg/ml) were common, and these isolates often displayed cross-resistance to hydrophobic antimicrobial agents (erythromycin and Triton X-100). Resistance to erythromycin and Triton X-100 is frequently due to overexpression of the mtrCDE-encoded efflux pump mediated by mutations in the mtrR gene, which encodes a transcriptional repressor that modulates expression of the mtrCDE operon. Accordingly, we questioned whether clinical isolates that express decreased azithromycin susceptibility harbor mtrR mutations. Promoter mutations that would decrease the level of expression of mtrR as well as a missense mutation at codon 45 in the mtrR-coding region that would result in a radical amino acid replacement within the DNA-binding motif of MtrR were found in these strains. When these mutations were transferred into azithromycin-susceptible strain FA19 by transformation, the susceptibility of gonococci to azithromycin was decreased by nearly 10-fold. The mtrCDE-encoded efflux pump system was responsible for this property since insertional inactivation of the mtrC gene resulted in enhanced susceptibility of gonococci to azithromycin. We conclude that the mtrCDE-encoded efflux pump can recognize azithromycin and that the emergence of gonococcal strains with decreased susceptibility to azithromycin can, in part, be explained by mtrR mutations.

Anti-Bacterial Agents↗

PubMed1998

Modulation of Neisseria gonorrhoeae susceptibility to vertebrate antibacterial peptides due to a member of the resistance/nodulation/division efflux pump family.

We have previously described the antibacterial capacity of protegrin-1 (PG-1), a cysteine-rich, cationic peptide from porcine leukocytes, against Neisseria gonorrhoeae. We now report genetic and biochemical evidence that gonococcal susceptibility to the lethal action of PG-1 and other structurally unrelated antibacterial peptides, including a peptide (LL-37) that is expressed constitutively by human granulocytes and testis and inducibly by keratinocytes, is modulated by an energy-dependent efflux system termed mtr. These results indicate that such efflux systems may enable mucosal pathogens like gonococci to resist endogenous antimicrobial peptides that are thought to act during infection.

Amino Acid Sequence↗

PubMed1997

Involvement of the gonococcal MtrE protein in the resistance of Neisseria gonorrhoeae to toxic hydrophobic agents.

Low-level resistance of Neisseria gonorrhoeae to toxic hydrophobic agents (HAs), including some antibiotics, is chromosomally mediated via the multiple transferable resistance (mtr) efflux system. The gene encoding the 48:3 kDa outer-membrane protein MtrE, which is associated with the mtr phenotype, was identified and is homologous to export-associated outer-membrane proteins, including the OprM (formerly OprK) lipoprotein of Pseudomonas aeruginosa. Insertional inactivation of the mtrE gene in N. gonorrhoeae strain FA19 resulted in the loss o the outer-membrane protein, with concomitant hypersusceptibility of the mutant strain to a range of HAs. The properties of this mutant confirmed the role of MtrE in multidrug resistance mediated by an active efflux mechanism. Secondary structure predictions for MtrE indicated a largely hydrophilic protein with a single alpha-helical transmembrane region. A transposon-like element, similar to that found downstream of the region containing the promoters for mtrR and mtrC in Neisseria meningitidis, was identified 63 bp downstream of the mtrE gene.

Amino Acid Sequence↗

PubMed1997

Protegrin structure and activity against Neisseria gonorrhoeae.

Protegrin 1 (PG-1) is a broad-spectrum antimicrobial peptide that contains 18 amino acid residues (RG GRLCYCRRRFCVCVGR) and has two intramolecular cystine disulfide bonds. To determine the minimal structure responsible for protegrin-mediated activity against Neisseria gonorrhoeae, we synthesized 15 protegrin variants and tested them against two well-characterized gonococcal strains. The MICs of PG-1 were 0.61 microM (1.31 microg/ml) for the serum-sensitive strain F 62 and 0.98 microM (2.11 microg/ml) for the serum-resistant strain FA 19. Six amino acid residues (Arg1, Gly2, Gly3, Arg4, Gly17, and Arg18) and either disulfide bond could be deleted from PG-1 without impairing its potency against strain F 62. In contrast, only Gly17 and Arg18 could be removed without decreasing its activity against FA 19. Protegrin congener 64a (PC-64a; LTYCRRRFCVTV), a variant of PG-1 with 12 amino acid residues and one disulfide bond, displayed MICs of 0.45 microM (0.68 microg/ml) for strain F 62 and 1.37 microM (2.07 microg/ml) for strain FA 19, which approximated those of intact PG-1. Serum-sensitive sac-1+ and sac-3+ transformants of N. gonorrhoeae FA 19 and two FA 19 derivatives with truncated lipooligosaccharide structures were more susceptible to PG-1 and variants with altered disulfide structures. These data suggest that structurally simpler protegrin variants, such as PC-64a, could be used as topical microbicides for N. gonorrhoeae. They also suggest that the cystine-stabilized antiparallel beta-sheet formed by PG-1 residues 5 to 16 is principally responsible for its activity against gonococci.

Amino Acid Sequence↗

PubMed1997

The MtrR repressor binds the DNA sequence between the mtrR and mtrC genes of Neisseria gonorrhoeae.

Gonococcal resistance to antimicrobial hydrophobic agents (HAs) is due to energy-dependent removal of HAs from the bacterial cell by the MtrCDE membrane-associated efflux pump. The mtrR (multiple transferrable resistance Regulator) gene encodes a putative transcriptional repressor protein (MtrR) believed to be responsible for regulation of mtrCDE gene expression. Gel mobility shift and DNase I footprint assays that used a maltose-binding protein (MBP)-MtrR fusion protein demonstrated that the MtrR repressor is capable of specifically binding the DNA sequence between the mtrR and mtrC genes. This binding site was localized to a 26-nucleotide stretch that includes the promoter utilized for mtrCDE transcription and, on the complementary strand, a 22-nucleotide stretch that contains the -35 region of the mtrR promoter. A single transition mutation (A-->G) within the MtrR-binding site decreased the affinity of the target DNA for MtrR and enhanced gonococcal resistance to HAs when introduced into HA-susceptible strain FA19 by transformation. Since this mutation enhanced expression of the mtrCDE gene complex but decreased expression of the mtrR gene, the data are consistent with the notion that MtrR acts as a transcriptional repressor of the mtrCDE efflux pump protein genes.

Bacterial Proteins↗

PubMed1996

Generation of antiserum to specific epitopes.

The ability to prevent disease by immunization with subunit vaccines that incorporate specific epitopes was demonstrated by DiMarchi et al. (1), who used a synthetic peptide to protect cattle against foot-and-mouth disease. However, generation of antibody to peptide antigens is often difficult owing to the small molecular mass and limited chemical complexity. We tested the hypothesis that recombinant DNA and synthetic peptide techniques would make it possible to stimulate vigorous immune responses to specific epitopes of an outer membrane protein of Neisseria gonorrhoeae. The MtrC AP1 sequence from the invariant MtrC gonococcal lipoprotein was genetically fused to maltose binding protein. The resultant fusion protein was used as the primary immunogen to stimulate MtrC AP1-specific antiserum. To enhance antibody production specific to MtrC AP1, boosting immunizations were performed with synthetic MtrC AP1 sequence contained in a multiple antigenic peptide system immunogen. The MtrC AP1-specific antiserum strongly recognized the MtrC protein on Western blots and appeared to bind native MtrC protein in situ. The generation of antibody in this fashion provides the technology to produce antibody to defined epitopes of any protein, including those found in the gonococcal outer membrane. The ability of those antibodies to inhibit bacterial growth or to activate complement protein can then be tested.

Amino Acid Sequence↗

PubMed1996

Bactericidal activity of a synthetic peptide (CG 117-136) of human lysosomal cathepsin G is dependent on arginine content.

The importance of individual amino acids in mediating the broad-spectrum bactericidal action of a 20-mer amphipathic, cationic peptide (CG 117-136) of human lysosomal cathepsin G was determined by using a single amino acid replacement strategy. This strategy revealed an important role for arginine because loss of any of the four arginine residues in CG 117-136 due to substitution with alanine, citrulline, or lysine residues resulted in a reduction of its bactericidal activity against both Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 33593. However, the replacement of a single alanine residue in CG 117-136 with arginine, but not glutamic acid, enhanced the activity of CG 117-136 against both P. aeruginosa and S. aureus. The importance of certain bulky, nonpolar amino acids for the bactericidal activity of CG 117-136 was also evident, since their substitutions by alanine diminished bactericidal activity. Accordingly, contributions of hydrophobic amino acids and structural considerations of the guanidinium side chain of arginine are major determinants in the broad-spectrum antimicrobial action of CG 117-136.

Amino Acid Sequence↗

PubMed1996

Susceptibility of Neisseria gonorrhoeae to protegrins.

We developed a sensitive and quantitative radial diffusion method to ascertain the susceptibility of six strains of Neisseria gonorrhoeae to antimicrobial peptides derived from mammalian leukocytes. The test organisms included the well-characterized serum-resistant FA19 and serum-sensitive F62 strains plus four antibiotic-resistant clinical isolates. Although each N. gonorrhoeae strain was resistant to human neutrophil defensins, all six were exquisitely sensitive to protegrins, a family of small beta-sheet antimicrobial peptides recently identified in porcine leukocytes. Protegrin-treated N. gonorrhoeae became vacuolated and had striking membrane changes when viewed by transmission and scanning electron microscopy. Because low concentrations of protegrins can also inactivate Chlamydia trachomatis and human immunodeficiency virus, they show promise for development as topical agents to avert sexually transmitted diseases.

Amino Acid Sequence↗

PubMed1995

Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux system.

The mtr (multiple transferable resistance) system of Neisseria gonorrhoeae determines levels of gonococcal resistance to hydrophobic agents (HAs), including detergent-like fatty acids and bile salts that bathe certain mucosal surfaces. The genetic organization of the mtr system was determined and found to consist of the mtrR gene, which encodes a transcriptional regulator (MtrR), and three tandemly linked genes termed mtrCDE. The mtrCDE genes were organized in the same apparent transcriptional unit, upstream and divergent from the mtrR gene. The mtrCDE-encoded proteins of N. gonorrhoeae were analogous to a family of bacterial efflux/transport proteins, notably the MexABOprK proteins of Pseudomonas aeruginosa and the AcrAE and EnvCD proteins of Escherichia coli, that mediate resistance to drugs, dyes, and detergents. Inactivation of the mtrC gene resulted in loss of the MtrC lipoprotein and rendered gonococci hypersusceptible to structurally diverse HAs; this revealed the importance of the mtr system in determining HAR in gonococci. Further support for a role of the mtrCDE gene complex in determining levels of HAR in gonococci was evident when transformants bearing mutations in the mtrR gene were analysed. In this respect, missense and null mutations in the mtrR gene were found to result in increased levels of MtrC and HAR. However, high levels of MtrC and HAR, similar to those observed for clinical isolates, were associated with a single bp deletion in a 13 bp inverted repeat sequence that intervened the divergent mtrR and mtrC genes. We propose that the 13 bp inverted-repeat sequence represents a transcriptional control element that regulates expression of the mtrRCDE gene complex, thereby modulating levels of gonococcal susceptibility to HAs.

Amino Acid Sequence↗