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PubMed2001

Genetic basis for activity differences between vancomycin and glycolipid derivatives of vancomycin.

Small molecules that affect specific protein functions can be valuable tools for dissecting complex cellular processes. Peptidoglycan synthesis and degradation is a process in bacteria that involves multiple enzymes under strict temporal and spatial regulation. We used a set of small molecules that inhibit the transglycosylation step of peptidoglycan synthesis to discover genes that help to regulate this process. We identified a gene responsible for the susceptibility of Escherichia coli cells to killing by glycolipid derivatives of vancomycin, thus establishing a genetic basis for activity differences between these compounds and vancomycin.

Anti-Bacterial Agents↗
PubMed2007

Sequencing and analysis of the biosynthetic gene cluster of the lipopeptide antibiotic Friulimicin in Actinoplanes friuliensis.

Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin, which is a cyclic peptide with one exocyclic amino acid linked to a branched-chain fatty acid acyl residue. The structural relationship to daptomycin and the excellent antibacterial performance of friulimicin make the antibiotic an attractive drug candidate. The complete friulimicin biosynthetic gene cluster of 24 open reading frames from A. friuliensis was sequenced and analyzed. In addition to genes for regulation, self-resistance, and transport, the cluster contains genes encoding peptide synthetases, proteins involved in the synthesis and linkage of the fatty acid component of the antibiotic, and proteins involved in the synthesis of the nonproteinogenic amino acids pipecolinic acid, methylaspartic acid, and 2,3-diaminobutyric acid. By using heterologous gene expression in Escherichia coli, we provide biochemical evidence for the stereoselective synthesis of L-pipecolinic acid by the deduced protein of the lysine cyclodeaminase gene pip. Furthermore, we show the involvement of the dabA and dabB genes in the biosynthesis of 2,3-diaminobutyric acid by gene inactivation and subsequent feeding experiments.

Aminobutyrates↗
PubMed1996

Antibacterial activity of a synthetic peptide (PR-26) derived from PR-39, a proline-arginine-rich neutrophil antimicrobial peptide.

PR-39 is a proline-arginine-rich (PR) neutrophil antibacterial peptide originally identified and purified from the porcine small intestine. We report on the synthesis of a functional antibacterial domain of PR-39, the first 26 amino acid residues of the NH2 terminus. PR-26 was as potent as or more potent than PR-39 against enteric gram-negative bacteria. This truncated form of PR-39 potentiated neutrophil phagocytosis of Salmonella choleraesuis and decreased the level of S. typhimurium invasion into intestinal epithelial cells. Scanning electron microscopy confirmed that these peptides did not lyse cells by pore-forming mechanisms; however, they potentiated the antibacterial capabilities of a pore-forming peptide, magainin A. In addition, PR-26 was not toxic to epithelial cells at concentrations several times greater than its bactericidal concentration. These data suggest that PR-39 and its functional domain, PR-26, may potentiate the host's defense capabilities against gram-negative infections.

Amino Acid Sequence↗
PubMed1996

Penicillin-binding protein 4 overproduction increases beta-lactam resistance in Staphylococcus aureus.

The Staphylococcus aureus mutant strain PVI selected in vitro for methicillin resistance overexpressed penicillin-binding protein (PBP) 4. In the wild-type parent strain the pbp4 gene was separated by 419 nucleotides from a divergently transcribed abcA locus coding for an ATP-binding cassette transporter. The mutant PVI was shown to have a deletion in the pbp4-abcA promoter region that affected pbp4 transcription but not expression of abcA. Introduction of the pbp4 gene plus the mutant promoter region into different genetic backgrounds revealed that PBP 4 overproduction was sufficient to increase in vitro-acquired methicillin resistance independently of other chromosomal genes. The role of the AbcA transporter in methicillin resistance remained unknown.

ATP-Binding Cassette Transporters↗
PubMed2003

Aminoglycoside efflux in Pseudomonas aeruginosa: involvement of novel outer membrane proteins.

The expression of tripartite multidrug efflux pumps such as MexA-MexB-OprM in Pseudomonas aeruginosa contributes to intrinsic resistance to a wide variety of antimicrobials, including beta-lactams, chloramphenicol, macrolides, quinolones, and tetracycline. The MexX-MexY linker-pump combination has been shown to be involved in intrinsic resistance to aminoglycosides, but the identity of the cognate outer membrane channel component remains under debate. Fourteen uncharacterized OprM homologs identified in the genome of P. aeruginosa were examined as candidates for this role by assessing the minimum inhibitory concentrations (MICs) of aminoglycosides in P. aeruginosa strain PAK knockout mutants lacking the corresponding genes. Insertional inactivation of OpmG, OpmI, and OpmH resulted in decreases of various degrees in the MICs of streptomycin, kanamycin, and gentamicin. When reintroduced into P. aeruginosa on multicopy plasmids, OpmG was able to complement the susceptibility of an opmG::miniTn5 mutant; however, cloned opmH, the proposed ortholog of Escherichia coli tolC according to our phylogenetic analysis, was able to only partially complement the opmH::miniTn5 mutant. Mini-microarray hybridization analysis demonstrated that opmG disruption does not affect expression of OpmI or OpmH (ruling out such indirect effects on aminoglycoside resistance); however, opmH disruption did have possible effects on expression of OpmG and OpmI. Based on the data, we propose that OpmG is a major outer membrane efflux channel involved in aminoglycoside efflux in P. aeruginosa PAK and that OpmI, its most related paralog, may share an overlapping function.

Aminoglycosides↗
PubMed2005

Genes involved in formation and attachment of a two-carbon chain as a component of eurekanate, a branched-chain sugar moiety of avilamycin A.

Eurekanate belongs to the important class of branched-chain carbohydrates present in a wide variety of natural sources. It is a component of avilamycin A, a potent inhibitor of bacterial protein synthesis targeting the 50S ribosomal subunit. The present work provides experimental proof for the function of two genes of the avilamycin biosynthetic gene cluster, aviB1 and aviO2, that are both involved in avilamycin structure modification. The functions of both genes were identified by gene inactivation experiments and nuclear magnetic resonance analyses of extracts produced by the mutants. We suggest that both AviO2 and AviB1 are involved in the biosynthesis of eurekanate within avilamycin biosynthesis. Moreover, two other genes (aviO1 and aviO3) have been inactivated, resulting in a breakdown of avilamycin production in the mutants ITO1 and ITO3, which clearly shows the essential role of both enzymes in avilamycin biosynthesis. The exact functions of both aviO1 and aviO3 remained unknown.

Bacillus subtilis↗
PubMed1991

Tet protein domains interact productively to mediate tetracycline resistance when present on separate polypeptides.

Both domains, alpha and beta, of the cytoplasmic membrane-localized Tet proteins encoded by the tet gene family (classes A through E) are required for resistance to tetracycline (Tcr) in gram-negative bacteria. Two inactive proteins, each containing a mutation in the opposite domain, are capable of complementation to produce Tcr. Similarly, inactive hybrid proteins expressed by interdomain gene hybrids constructed between tet(B) and tet(C) [tet(B) alpha/(C) beta and tet(C) alpha/(B) beta] together produce significant Tcr via trans complementation (R.A. Rubin and S. B. Levy, J. Bacteriol. 172:2303-2312, 1990). A derivative of tet(B) was constructed to express the two domains of Tet(B) as separate polypeptides, neither containing intact the central, hydrophilic interdomain region. Cells harboring this tet(B) mutant expressed Tcr at about 20% the level conferred by intact tet(B). As expected, no detectable amount of a full-length Tet protein was expressed. A polypeptide corresponding to the alpha domain was observed. Interdomain hybrids between tet(B) and tet(C) containing a frameshift at the fusion junction, designed to result in expression of each of the four domains on separate polypeptides, showed trans complementation without production of detectable full-length proteins. Levels of Tcr were greater than or equal to those previously observed in complementations using full-length hybrid proteins. These results strongly suggest that polypeptides harboring individual alpha and beta domains, lacking an intact interdomain region, can interact productively in the cell to confer Tcr.

Amino Acid Sequence↗
PubMed1995

Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system.

The coding sequence of the Haemophilus influenzae ORF I gene was amplified by PCR and cloned into different Escherichia coli expression vectors. The ORF I-encoded protein was approximately 90 kDa and bound 3H-benzyl-penicillin and 125I-cephradine. This high-molecular-weight penicillin-binding protein (PBP) was also shown to possess transglycosylase activity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli delta ponA ponB::spcr cells in transcomplementation experiments, establishing the functional relevance of the significant amino acid homology seen between E. coli PBP 1A and 1B and the H. influenzae ORF I product. In addition, the physiological functioning of the H. influenzae ORF I (PBP 1A) product in a heterologous species established the ability of the enzyme not only to recognize the E. coli substrate but also to interact with heterologous cell division proteins. The affinity of the ORF I product for 3H-benzylpenicillin and 125I-cephradine, the MIC of beta-lactams for E. coli delta ponA ponB::spcr expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molecular-weight PBPs group the ORF I protein into the PBP 1A family of high-molecular-weight PBPs.

Bacterial Proteins↗
PubMed1998

NADH dehydrogenase defects confer isoniazid resistance and conditional lethality in Mycobacterium smegmatis.

Isoniazid (INH) is a highly effective drug used in the treatment and prophylaxis of Mycobacterium tuberculosis infections. Resistance to INH in clinical isolates has been correlated with mutations in the inhA, katG, and ahpC genes. In this report, we describe a new mechanism for INH resistance in Mycobacterium smegmatis. Mutations that reduce NADH dehydrogenase activity (Ndh; type II) cause multiple phenotypes, including (i) coresistance to INH and a related drug, ethionamide; (ii) thermosensitive lethality; and (iii) auxotrophy. These phenotypes are corrected by expression of one of two enzymes: NADH dehydrogenase and the NADH-dependent malate dehydrogenase of the M. tuberculosis complex. The genetic data presented here indicate that defects in NADH oxidation cause all of the mutant traits and that an increase in the NADH/NAD+ ratio confers INH resistance.

Amino Acid Sequence↗
PubMed2004

Clinical isolates of Staphylococcus intermedius masquerading as methicillin-resistant Staphylococcus aureus.

Staphylococcus intermedius is a zoonotic organism that can be associated with human disease. We report two separate cases of S. intermedius infection in which a false-positive rapid penicillin binding protein 2a latex test in conjunction with the phenotypic properties of beta-hemolysis and coagulase positivity allowed the clinical isolates to masquerade as methicillin-resistant Staphylococcus aureus. 16S rRNA gene sequencing and the absence of mecA revealed the strains to be methicillin-susceptible S. intermedius.

Adult↗
PubMed1997

[Significance of the detection of fimbriae protein type adhesins on bacteria isolated in acute pyelonephritis in children].

A prospective bacteriological study in 50 children with acute pyelonephritis (APN) (32 girls and 18 boys) and 132 children with lower urinary tract infections (LUTI) (89 girls and 43 boys) was conducted from May to December 1993. Infection was defined by Kass' criteria and APN was defined by the clinical findings. C-Reactive Protein (CRP) assay and postcontrast computed tomography in the presence of a doubt concerning the diagnosis. Escherichia coli (EC) was the bacterial species most frequently isolated (76%). A systematic search for fimbriae protein adhesins (group PAP: pyelonephritis associated pil) on the EC was performed by haemagglutination (human group A red blood cells). 64% of EC possessed fimbriae protein adhesions in the APN group versus only 20% in the LUTI group. In children in whom an organic abnormality was demonstrated, the incidence of fimbriae protein-positive EC was 33% while in children with no organic abnormality, particularly without reflux, 89% of EC presented fimbriae protein. A statistically significant difference was demonstrated between these two groups (p < 0.01). The results of this study illustrate the important role of these adhesins in the development of APN. These adhesins facilitate countercurrent ascension of bacteria in the ureter towards the upper urinary tract and can make the bacteria resistant to certain antibiotics. Testing for fimbriae protein can be useful in clinical practice when investigating the aetiology of APN in the absence of demonstrated reflux. A latex test should soon be available to facilitate the detection of fimbriae protein.

Acute Disease↗
PubMed1998

Active surveillance of meningococcal meningitis in Poland.

Starting from 1970, the notification of N. meningitidis cases in Poland was compulsory and separated from other cases of meningitis purulenta. Based on the experience of European Monitoring Group on Meningococci, the active surveillance of meningococcal meningitis in Poland was initiated in April 1995. It was the first time that such study was conducted to recognise the actual situation of meningococcal meningitis infections in our country. Ninety seven N. meningitidis strains were isolated (31 in 1995 and 66 in 1996) from cerebrospinal fluid (CSF) of meningitis patients hospitalized in 54 hospitals located in 33 out of 49 provinces of Poland. Most patients were below 2 years of age and 43% belonged to infant group. Meningococcal strains were phenotypically characterized as follow: identification of N. meningitidis was performed by Gram staining, oxidase and catalase tests as well as latex or diagnostic sera agglutination assays. Meningococcal serotypes and subtypes were determined by whole-cell ELISA with monoclonal antibodies. The predominant meningococcal serogroup during 1995 and 1996 was B (80% of all isolates tested), the serogroup C (12.6%) and W-135 (3.5%). Only two non-groupable and two serogroup A strains were isolated in Poland. Active surveillance allowed to determine B:22:P1.14 to be the most prevalent N. meningitidis phenotype in Poland. Two isolates of N. meningitidis phenotype C:2a:P1.2,5, which caused emergency situation in Czech Republic since 1993, were isolated from CSF of patients in October 1996 in southern Poland. All strains were susceptible to cefotaxime, chloramphenicol, ciprofloxacin, rifampin and tetracycline; some strains were resistant to sulphonamides (60.6% - MIC = 32 mg/l and 14.8% - MIC = 128 mg/l). Only one of the tested strains in two years surveillance study in Poland was resistant to penicillin (MIC = 2 mg/l).

Adolescent↗
PubMed1986

[A study by the National Reference Center for Antibiotics on inocula for antibiotic sensitivity testing].

We compared the diameters of inhibition zones obtained during antibiotic sensitivity testing using two different techniques for preparing and seeding the inoculum, i.e. photometric adjustment followed by flooding, and turbidity adjustment followed by swab streaking. There was no significant difference between the results recorded following photometry-flooding of a light inoculum (2 to 3 X 10(6) CFU/ml) and following swab seeding of a bacterial suspension with a turbidity equal to 0.5 Mac Farland unit. These results indicate that both methods tested answer the NCCLS performance standards for disc antibiotic sensitivity testing and the critical values of the S.F.M. Antibiotic Sensitivity Testing Committee.

Escherichia coli↗
PubMed2000

Differential growth inhibition as a tool to increase the discriminating power of killer toxin sensitivity in fingerprinting of yeasts.

A panel of 27 cell-free crude killer toxin preparations were used in fingerprinting 45 Saccharomyces cerevisiae and 11 Saccharomyces exiguus strains. The differential sensitivity to different mycocins was evaluated both as binary data matrix (presence-absence of killing effect), and by considering the growth inhibition areas (measured by agar diffusion well bioassay). The first approach gave an individual fingerprinting of 68% of sensitive strains, whereas the second gave a total and reproducible (P<0.01) discrimination of all tested strains.

Antibiosis↗