Re-evaluation of the significance of penicillin binding protein 3 in the susceptibility of Listeria monocytogenes to ß-lactam antibiotics.

BACKGROUND: Penicillin binding protein 3 (PBP3) of L. monocytogenes has long been thought of as the primary lethal target for ß-lactam antibiotics due to the excellent correlation between the MICs of different ß-lactams and their affinity for this protein. The gene encoding PBP3 has not yet been directly identified in this gram-positive bacterium, but based on in silico analysis, this protein is likely to be encoded by lmo1438. However, studies examining the effects of mutations in genes encoding known and putative L. monocytogenes PBPs have demonstrated that inactivation of lmo1438 does not affect sensitivity to ß-lactams. RESULTS: In this study, overexpression of lmo1438 was achieved using an inducible (nisin-controlled) expression system. This permitted the direct demonstration that lmo1438 encodes PBP3. PBP3 overexpression was accompanied by slightly elevated PBP4 expression. The recombinant strain overexpressing PBP3 displayed significant growth retardation and greatly reduced cell length in the stationary phase of growth in culture. In antibiotic susceptibility assays, the strain overexpressing PBP3 displayed increased sensitivity to subinhibitory concentrations of several ß-lactams and decreased survival in the presence of a lethal dose of penicillin G. However, the MIC values of the tested ß-lactams for this recombinant strain were unchanged compared to the parent strain. CONCLUSIONS: The present study allows a reevaluation of the importance of PBP3 in the susceptibility of L. monocytogenes to ß-lactams. It is clear that PBP3 is not the primary lethal target for ß-lactams, since neither the absence nor an excess of this protein affect the susceptibility of L. monocytogenes to these antibiotics. The elevated level of PBP4 expression observed in the recombinant strain overexpressing PBP3 demonstrates that the composition of the L. monocytogenes cell wall is subject to tight regulation. The observed changes in the morphology of stationary phase cells in response to PBP3 overexpression suggests the involvement of this protein in cell division during this phase of growth.

An update on some structural aspects of the mighty miniwall.

Peptidoglycan (PG), the mighty miniwall, is the main structural component of practically all bacterial cell envelopes and has been the subject of a wealth of research over the past 60 years, if only because its biosynthesis is the target of many antibiotics that have successfully been used in the treatment of bacterial infections. This review is mainly focused on the most recent achievements in research on the modification of PG glycan strands, which contribute to the resistance of bacteria to the host immune response to infection and to their own lytic enzymes, and on studies on the spatial organization of the macromolecule.

Identification of the full set of Listeria monocytogenes penicillin-binding proteins and characterization of PBPD2 (Lmo2812).

BACKGROUND: Bacterial penicillin-binding proteins (PBPs) can be visualized by their ability to bind radiolabeled or fluorescent ß-lactam derivatives both whole cells and membrane/cell enriched fractions. Analysis of the Listeria monocytogenes genome sequence predicted ten genes coding for putative PBPs, but not all of their products have been detected in studies using radiolabeled antibiotics, thus hindering their characterization. Here we report the positive identification of the full set of L. monocytogenes PBPs and the characteristics of the hitherto undescribed PBPD2 (Lmo2812). RESULTS: Eight L. monocytogenes PBPs were identified by the binding of fluorescent ß-lactam antibiotic derivatives Boc-FL, Boc-650 and Amp-Alexa430 to proteins in whole cells or membrane/cell wall extracts. The gene encoding a ninth PBP (Lmo2812) was cloned and expressed in Escherichia coli as a His-tagged protein. The affinity purified recombinant protein had DD-carboxypeptidase activity and preferentially degraded low-molecular-weight substrates. L. monocytogenes mutants lacking the functional Lmo2812 enzyme were constructed and, compared to the wild-type, the cells were longer and slightly curved with bent ends.Protein Lmo1855, previously designated PBPD3, did not bind any of the antibiotic derivatives tested, similarly to the homologous enterococcal protein VanY. CONCLUSIONS: Nine out of the ten putative L. monocytogenes PBP genes were shown to encode proteins that bind derivatives of ß-lactam antibiotics, thus enabling their positive identification. PBPD2 (Lmo2812) was not visualized in whole cell extracts, most probably due to its low abundance, but it was shown to bind Boc-FL after recombinant overexpression and purification. Mutants lacking Lmo2812 and another low molecular mass (LMM) PBP, PBP5 (PBPD1)--both with DD-carboxypeptidase activity--displayed only slight morphological alterations, demonstrating that they are dispensable for cell survival and probably participate in the latter stages of peptidoglycan synthesis. Since Lmo2812 preferentially degrades low-molecular- mass substrates, this may indicate a role in cell wall turnover.

Simultaneous degradation of waste phosphogypsum and liquid manure from industrial pig farm by a mixed community of sulfate-reducing bacteria.

The utilization of pig manure as a source of nutrients for the dissimilatory reduction of sulfates present in phosphogypsum was investigated. In both types of media used (synthetic medium and raw pig manure) increased utilization of sulfates with growing COD/SO4(2-)ratio in the medium was observed. The percent of sulfate reduction obtained in synthetic medium was from 18 to 99%, whereas the value for cultures set up in raw liquid manure was from 12% (at COD/SO4(2-) of 0.3) up to as high as 98% (at COD/SO4(2-) equal 3.80). Even with almost complete reduction of sulfates the percent of COD reduction did not exceed 55%. Based on the results obtained it was concluded that the effectiveness of removal of sulfates and organic matter by sulfate-reducing bacteria (SRB) depends to a considerable degree on the proportion between organic matter and sulfates in the purified wastewaters. The optimal COD/SO4(2-)ratio for the removal oforganic matter was between 0.6 and 1.2 whereas the optimal ratio for the removal of sulfates was between 2.4 and 4.8.

Inactivation of the wall-associated de-N-acetylase (PgdA) of Listeria monocytogenes results in greater susceptibility of the cells to induced autolysis.

Several species of Gram-positive bacteria have cell wall peptidoglycan (syn. murein) in which not all of the sugar moieties are N-acetylated. This has recently been shown to be a secondary effect, caused by the action of a peptidoglycan N-acetylglucosamine deacetylase. We have found that the opportunistic pathogen Listeria monocytogenes is unusual in having three enzymes with such activity, two of which remain in the cytoplasm. Here, we examine the enzyme (PgdA) that crosses the cytoplasmic membrane and is localized in the cell wall. We purified a hexa-His-tagged form of PgdA to study its activity and constructed a mutant devoid of functional Lmo0415 (PgdA) protein. L. monocytogenes PgdA protein exhibited peptidoglycan N-acetylglucosamine deacetylase activity with natural substrates (peptidoglycan) from both L. monocytogenes and Escherichia coli as well as the peptidoglycan sugar chain component N-acetylglucosamine, but not with N-acetylmuramic acid. As was reported recently [6], inactivation of the structural gene was not lethal for L. monocytogenes nor did it affect growth rate or morphology of the cells. However, the pgdA mutant was more prone to autolysis induced by such agents as Triton X-100 and EDTA, and is more susceptible to the cationic antimicrobial peptides (CAMP) lysozyme and mutanolysin, using either peptidoglycan muramidases or autolysis-inducing agents. The pgdA mutant was also slightly more susceptible than the wild-type strain to the action of certain beta-lactam antibiotics. Our results indicate that protein PgdA plays a protective physiological role for listerial cells.

Characterization of Listeria monocytogenes protein Lmo0327 with murein hydrolase activity.

Listeria monocytogenes is an ubiquitous gram-positive, opportunistic food-borne human and animal pathogen. To date, five L. monocytogenes autolysins have been characterized: p60, p45, Ami, MurA and Auto and the preliminary results of our studies show that FlaA, a flagellar protein of L. monocytogenes, also has murein-degrading activity. In this study, a gene coding a 144 kDa protein (Lmo0327) with murein hydrolase activity was identified from a lambda Zap expression library of L. monocytogenes EGD genomic DNA, using a direct screening protocol involving the plating of infected Escherichia coli XL1-blue MRF' cells onto medium containing Bacillus subtilis murein, a substrate for autolytic proteins. Protein Lmo0327 has a signal sequence, a N-terminal LRR domain and a C-terminal wall-anchoring LPXTG motif. In order to examine the roles of this enzyme and the putative transcription regulator coded by gene lmo0326 located upstream of lmo0327, both structural genes were insertionally inactivated by site-specific integration of a temperature-sensitive plasmid. We show that Lmo0327 is a surface protein covalently linked to murein and that the putative transcription regulator Lmo0326 can be assumed to positively regulate the expression of gene lmo0327. The enzyme, which we have shown to have murein-hydrolysing activity, plays a role in cell separation and murein turnover.

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes.

Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M(-1) s(-1) from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to beta-lactam antibiotics but did increase the resistance of the mutant to moenomycin.

Susceptibility of Listeria monocytogenes strains isolated from dairy products and frozen vegetables to antibiotics inhibiting murein synthesis and to disinfectants.

The susceptibility of 96 strains of Listeria monocytogenes isolated from food to antibiotics and disinfectants currently used in human therapy, veterinary, medicine and food industry was determined by a standard operating procedure--broth dilution method. Antimicrobial agents included the beta-lactams ampicillin and penicillin, the lantibiotic nisin, and the disinfectants benzalkonium chloride and chlorhexidine gluconate. Among the studied strains we found 13 strains with 8-fold, 7 strains with 16-fold and 2 strains with 32-fold decreased susceptibility to ampicillin, as determined by MIC, compared to wild type reference strain. Interestingly, the mentioned strains were isolated from frozen vegetables and soups, none of the isolates from dairy products showed any elevated resistance to the studied antimicrobial agents. The occurrence in food products of strains with increased resistance to ampicillin is disquieting, especially since 3-lactams are the most frequent antibiotic of choice in the therapy of infections caused by the pathogen.

Listeria monocytogenes EGD lacking penicillin-binding protein 5 (PBP5) produces a thicker cell wall.

We report on the cloning of the structural gene for penicillin-binding protein 5 (PBP5), lmo2754. We also describe the enzymatic activity of PBP5 and characterize a mutant lacking this activity. Purified PBP5 has dd-carboxypeptidase activity, removing the terminal D-alanine residue from murein pentapeptide side chains. It shows higher activity against low molecular weight monomeric pentapeptide substrates compared to dimeric pentapeptide compound. Similarly, PBP5 preferentially cleaves monomeric pentapeptides present in high-molecular weight murein sacculi. A Listeria monocytogenes mutant lacking functional PBP5 was constructed. Cells of the mutant are viable, showing that the protein is dispensable for growth, but grow slower and have thickened cell walls.

Analysis of the murein of a Listeria monocytogenes EGD mutant lacking functional penicillin binding protein 5 (PBP5).

Cells of a mutant of Listeria monocytogenes lacking functional PBP5, an enzyme with DD-carboxypeptidase activity, make thicker cells walls. In this study we show that the muropeptide profile of the mutant, obtained after HPLC analysis of a muramidase digest of cell wall murein, differs from that for the wild type strain. The main differences embrace strongly reduced disaccharide-tripeptide content, strongly increased amounts of pentapeptide-containing muropeptides and a shift in profile from less cross-linked muropeptides (monomers, dimers) towards more highly cross-linked ones.

Classes and functions of Listeria monocytogenes surface proteins.

Listeria monocytogenes is an opportunistic pathogen that causes infections collectively termed listeriosis, which are related to the ingestion of food contaminated with these gram-positive rods. The pathogenicity of L. monocytogenes is determined by the following virulence factors: listeriolysin O, protein ActA, two phospholipases C, internalins (In1A and In1B), protein CwhA and a metalloprotease. The bacterium is a model organism in studies on the pathogenesis of intracellular parasites. It is able to penetrate, multiply and propagate in various types of eukaryotic cells and is also able to overcome the three main barriers encountered in the host: the intestinal barrier, the blood-brain barrier and the placenta. Based on L. monocytogenes genome sequence analysis 133 surface proteins have been identified. In particular, the large number of proteins covalently bound to murein sets L. monocytogenes apart from other gram-positive bacteria. The ability of this pathogen to multiply in various environments as well as the possibility of its interaction with many kinds of eukaryotic cells is, in fact, made possible by the large number of surface proteins.

Mechanism of vancomycin resistance in methicillin resistant Staphylococcus aureus.

A collection of laboratory mutants and clinical MRSA strains, additionally exhibiting resistance to glycopeptide antibiotics, was studied in detail. The nature of resistance to glycopeptides was found to be different from that existing in vancomycin resistant (VR) enterococci. The mutants produced abnormal murein in which the level of highly oligomeric muropeptides was drastically reduced. Biochemical and genetic analyses of Penicillin Binding Proteins (PBPs) showed inactivation of PBP4. Changes in other PBPs were not apparent, except for PBP2a that was inactivated in the highly VR mutant VM. Transposon inactivation of the pbpB gene and several other genes involved in synthesis of staphylococcal peptidoglycan all caused dramatic reduction of glycopeptide resistance in the staphylococcal mutants. While inactivation of PBP2a slightly increased the levels of glycopeptide resistance, a combination of vancomycin or teicoplanin with beta-lactam inhibitors, chosen on the basis of their relatively selective affinities for individual staphylococcal PBPs completely inhibited the expression of glycopeptide resistance in MRSA. Glycopeptide antibiotics caused a virtually complete inhibition of cell wall turnover and autolysis and massive overgrowth of cell wall material in the glycopeptide resistant mutants. Bacteria were able to remove quantitatively glycopeptide molecules from the growth medium, and sequestered antibiotic could be recovered in biologically active form from the purified cell walls. These observations and the results of the vancomycin binding studies suggest alterations in the structural organization of the mutants' cell wall such that access of glycopeptide molecules to the sites of wall biosynthesis is blocked by steric hindrance.

Murein-hydrolyzing activity of flagellin FlaA of Listeria monocytogenes.

In this preliminary report we show that a 29 kDa surface protein of Listeria monocytogenes EGD removed from cells with 4 M LiCl has peptidoglycan (murein) hydrolyzing activity, as revealed by zymographic analysis using Bacillus subtilis murein and heat-killed Micrococcus luteus cells casted in the gel. Following two-dimensional electrophoresis, the protein was electroblotted to PVDF membrane and its identity (FlaA) was revealed by sequencing. Peptidoglycan hydrolysing activity of FlaA purified by FPLC on Mono-S Sepharose against labelled Escherichia coli murein was demonstrated.

Characteristics of bacterial strains inhabiting the wood of coniferous trees.

The presented studies embraced samples of wood chips from coniferous trees which contained layers of duramen, alburnum and bark. Microbiological analysis involved qualitative and quantitative determination of bacterial flora inhabiting the studied wood material. The wood chips were found to contain primarily species belonging to the genera Bacillus and Pseudomonas. The presence of the potentially pathogenic species Bacillus cereus 1, Sphingomonas paucimobilis, Aeromonas salmonicida and Chryseomonas luteola was also demonstrated.

Antimicrobial resistance of Listeria monocytogenes.

Listeria monocytogenes is an opportunistic pathogen that causes rare but frequently fatal infections, termed listerioses. In general, strains of L. monocytogenes are susceptible to a wide range of antibiotics, except for the cephalosporins, fluorochinolones and fosfomycin (Hof, 1991). The current therapy of choice is a combination of ampicillin and aminoglycoside, usually gentamicin (Lorber, 1997). In cases when it is not possible to use a beta-lactam antibiotic, second-choice therapy involves the use of an association of trimethoprim with a sulfonamide, such as in co-trimoxazole, in which the more active in the combination seems trimethoprim, synergized by the sulfa compound. Other second line agents for listeriosis include erythromycin and vancomycin (Temple and Nahata, 2000). The first strains of L. monocytogenes resistant to antibiotics were reported in 1988 (Poyart-Salmeron et al. 1990) The present paper reviews the current state of affairs with regard to the resistance of L. monocytogenes isolated from food products and clinical material to different antibiotics, with particular emphasis on those used in the therapy of listeriosis.

Ampicillin resistance in Listeria monocytogenes acquired as a result of transposon mutagenesis.

We have used plasmid pLTV3, which carries transposon Tn917, to obtain a series of mutants of Listeria monocytogenes EGD showing varied degrees of resistance to ampicillin and other beta-lactam antibiotics, including imipenem. In this paper we focus on the characteristics of two strains in which decreased susceptibility to ampicillin is accompanied by changes in the structure of the cell wall murein and cell-wall related changes of phenotype.

Tolerance to creosote oil of bacteria of the genus Pseudomonas isolated from the wood of coniferous trees.

A number of Pseudomonas sp. strains isolated from wood shavings not preserved with chemical agents were characterized by tolerance to concentrated creosote oil. Of eleven strains subjected to closer scrutiny, five showed good or very good growth in minimal medium with creosote oil as sole carbon and energy source. These isolates can be of potential use for the biodegradation of waste wood conserved with creosote oil.

Penicillin-binding proteins of listeria monocytogenes--a re-evaluation.

Intact Listeria monocytogenes cells or membranes isolated from them were treated with [3H]penicillin to allow identification of the penicillin binding proteins (PBPs) located in the cytoplasmic membrane. In the former case the PBPs were released from the cells following disruption of the cell wall murein with Listeria monocytogenes bacteriophage lysin. The procedure described by Dougherty et al. (1996) for Escherichia coli, with some modifications, was used to evaluate the M(r)s of the individual PBPs and allowed direct quantitation of their copy number.