Staphylococcus aureus Penicillin-Binding Protein 2 Can Use Depsi-Lipid II Derived from Vancomycin-Resistant Strains for Cell Wall Synthesis.

Vancomycin-resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide-containing modified cell-wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin-binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild-type S. aureus. However, it is unclear whether VRSA processes the depsipeptide-containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi-lipid I, a cell-wall precursor of VRSA. By using this chemistry, we prepared a depsi-lipid II analogue as substrate for a cell-free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi-lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin.

The Bacillus subtilis spoVD gene encodes a mother-cell-specific penicillin-binding protein required for spore morphogenesis.

The Bacillus subtilis spoVD gene has been cloned and sequenced. It encodes a 71,262 Da protein with extensive sequence similarity to penicillin-binding proteins from various organisms. The context of this gene in the B. subtilis chromosome, immediately upstream of the mur operon, suggests that it is related to the pbpB gene of Escherichia coli, which is involved in the synthesis of septal peptidoglycan during cell division. Expression of spoVD in E. coli leads to the synthesis of a membrane-associated protein of the size expected for SpoVD, which can bind labelled penicillin. However, insertional disruption of the spoVD gene has no effect on vegetative growth or division: a second pbp-like gene immediately upstream of spoVD is probably the functional homologue of E. coli pbpB. spoVD seems instead to have a specialized role in the morphogenesis of the spore cortex, which is a modified form of peptidoglycan. spoVD transcription appears to occur from a promoter recognized by the sigma E form of RNA polymerase. Analysis of the expression of a spoVD'-lacZ reporter gene supports this notion and indicates that a second level of negative regulation is dependent on the SpoIIID protein. SpoVD synthesis probably occurs only in the mother cell since both sigma E and SpoIIID are thought to be specific to this cell type. Such localization of SpoVD synthesis was supported by the results of a genetic test showing that expression of spoVD only in the mother cell is sufficient for spore formation. The results support the proposition that spore cortex formation is determined primarily by the mother cell.

Suppression of defective-sporulation phenotypes by mutations in transcription factor genes of Bacillus subtilis.

Mutations in the Bacillus subtilis major RNA polymerase sigma factor gene (rpoD/crsA47) and a sensory receiver gene (spoOA/rvtA11) are potent intergenic suppressors of several stage 0 sporulation mutations (spoOB, OE, OF & OK). We show here that these suppressors also rescue temperature-sensitive sporulation phenotypes (Spots) caused by mutations in RNA polymerase, ribosomal protein, and protein synthesis elongation factor EF-G genes. The effects of the crsA and rvtA suppressors on RNA polymerase and ribosomal protein spots mutations are similar to those previously described for mutations in another intergenic suppressor gene rev. We have examined the effects of rvtA and crsA mutations on the expression of sporulation-associated membrane proteins, including flagellin and penicillin binding protein 5* (PBP 5*). Both suppressors restored sporulation and synthesis of PBP 5* in several spoO mutants. However, only rvtA restored flagellin synthesis in spoO suppressed backgrounds. The membrane protein phenotypes resulting from the presence of crsA or rvtA suppressors in spoO strains suggests that these suppressors function via distinct molecular mechanisms. The rvtA and crsA mutations are also able to block the ability of ethanol to induce spoO phenocopies at concentrations of ethanol which prevent sporulation in wild type cells. The effects of ethanol on sporulation-associated membrane protein synthesis in wild type and suppressor containing strains have been examined.

Mechanisms of methicillin resistance in staphylococci.

The continuously high prevalence of methicillin-resistant staphylococci (MRS) throughout the world is a constant threat to public health, owing to the multiresistant characteristics of these bacteria. Methicillin resistance is phenotypically associated with the presence of the penicillin-binding protein 2a (PBP2a) not present in susceptible staphylococci. This protein has a low binding affinity for beta-lactam antibiotics. It is a transpeptidase which may take over cell wall synthesis during antibiotic treatment when normally occurring PBPs are inactivated by ligating beta-lactams. PBP2a is encoded by the mecA gene, which is located in mec, a foreign DNA region. Expression of PBP2a is regulated by proteins encoded by the plasmid-borne blaR1-bla1 inducer-repressor system and the corresponding genomic mecRl-mecl system. The blaRl-blal products are important both for the regulation of beta-lactamase and for mecA expression. Methicillin resistance is influenced by a number of additional factors, e.g. the products of the chromosomal fem genes which are important in the synthesis of normal peptidoglycan precursor molecules. Inactivation of fem-genes results in structurally deficient precursors which are not accepted as cell wall building blocks by the ligating PBP2a transpeptidase during antibiotic treatment. This may result in reduced resistance to beta-lactam antibiotics. Inactivation of genes affecting autolysis has shown that autolytic enzymes are also of importance in the expression of methicillin resistance. Methicillin resistance has evolved among earth microorganisms for protection against exogenous or endogenous antibiotics. Presumably the mec region was originally transferred from coagulase negative staphylococci (CNS) to Staphylococcus aureus (SA). A single or a few events of this kind with little subsequent interspecies transfer had been anticipated. However, recent data suggest a continuous horizontal acquisition by S. aureus of mec, being unidirectional from CNS to SA. Methicillin resistance may also be associated with mechanisms independent of mecA, resulting in borderline methicillin resistance. These mechanisms include beta-lactamase hyperproduction, production of methicillinases, acquisition of structurally modified normal PBPs, or the appearance of small colony variants of SA. Most MRS are multiresistant, and the mec region may harbour several resistance determinants, resulting in a clustering of resistance genes within this region.

Mechanisms of methicillin resistance in Staphylococcus aureus and methods for laboratory detection.

Three distinctly different mechanisms of methicillin resistance have been described in Staphylococcus aureus. The best-documented and probably most important mechanism is production of a unique, low affinity penicillin-binding protein, PBP 2a. Strains possessing PBP 2a are resistant to methicillin, oxacillin, and probably all other currently available beta-lactam antibiotics. Two additional mechanisms of reduced susceptibility to methicillin have been described. Borderline resistance (BORSA) to the semi-synthetic penicillins has been attributed to the hyperproduction of normal staphylococcal beta-lactamase. A third mechanism has recently been advanced that describes an intermediate level of resistance to methicillin due to production of modified, normal PBPs with reduced affinity for beta-lactams (MODSA). Little is known regarding the prevalence or clinical significance of the BORSA and MODSA strains. The most reliable in vitro susceptibility test methods for detecting MRSA (strains possessing PBP 2a) include the microdilution minimum inhibitory concentration (MIC) test (with 2% NaCl supplemented broth), the oxacillin agar screen plate test (incorporating 6 micrograms/ml oxacillin in 4% NaCl supplemented agar), and the National Committee for Clinical Laboratory Standards (NCCLS) disk diffusion test with oxacillin. All three methods use direct inoculum preparation and incubation of tests at 35 degrees C for a full 24 hours.

Effects of penicillin-binding protein 4 overproduction in Staphylococcus aureus.

The pbp4 gene of a Staphylococcus aureus strain selected stepwise in vitro for growth on increasing concentrations of penicillin and of its susceptible parent strain showed overall identity except in the promoter region. In the mutant a deletion upstream of the pbp4 structural gene removed 90 nucleotides (nt) that were framed by a 12 nt inverted repeat. This deletion occurred in step 4 of the in vitro selection procedure and was paralleled by a significant increase in the penicillin-binding protein 4 (PBP4) production. The in vitro step selected mutant showed a remarkable increase in the cross-linking of the peptidoglycan compared to its parent. This was linked to morphological changes in the appearance of the cells, which were surrounded by a very thick and fuzzy cell wall.

Intrinsic penicillin resistance in enterococci.

Penicillin resistance development in enterococci has been associated with overproduction of a low-affinity penicillin-binding protein (PBP) that is a normal component of the PBP pattern of these bacteria and is apparently able to substitute the functions of the other PBPs. In resistant mutants of Enterococcus hirae ATCC 9790 the low-affinity PBP (PBP5) overproduction was associated with a deletion in a genetic element, located 1 kb upstream of the pbp5 gene, which negatively controlled PBP5 synthesis. Hypersusceptibility to penicillin was associated with a point mutation in the pbp5 gene, which causes premature termination of translation. Structural homologies between low-affinity PBPs of the different enterococcal species have been suggested by cross-reactivity of antibodies raised against E. hirae PBP5 with PBP5 of Enterococcus faecium and Enterococcus faecalis. Acquisition of a high-level ampicillin resistance in E. faecium was associated with overproduction of PBP5, which, compared with PBP5 of moderately resistant strains, appeared to be modified in its penicillin-binding capability. The modified phenotype of PBP5 was found to be associated to some amino acid substitutions in the region between the SDN and KTG motifs. In particular, the substitution converting a polar residue (T) in a nonpolar one (A or I) could play an important role in remodeling the penicillin-binding domain and determining the decrease in penicillin affinity.

Spontaneous nisin-resistant Listeria monocytogenes mutants with increased expression of a putative penicillin-binding protein and their sensitivity to various antibiotics.

A concern regarding the use of bacteriocins, as for example the lantibiotic nisin, for biopreservation of certain food products is the possibility of resistance development and potential cross-resistance to antibiotics in the target organism. The genetic basis for nisin resistance development is as yet unknown. We analyzed changes in gene expression following nisin resistance development in Listeria monocytogenes 412 by restriction fragment differential display. The mutant had increased expression of a protein with strong homology to the glycosyltransferase domain of high-molecular-weight penicillin-binding proteins (PBPs), a histidine protein kinase, a protein of unknown function, and ClpB (putative functions from homology). The three former proteins had increased expression in a total of six out of 10 independent mutants originating from five different wild-type strains, indicating a prevalent nisin resistance mechanism under the employed isolation conditions. Increased expression of the putative PBP may affect the cell wall composition and thereby alter the sensitivity to cell wall-targeting compounds. The mutants had an isolate-specific increase in sensitivity to different beta-lactams and a slight decrease in sensitivity to another lantibiotic, mersacidin. A model incorporating these observations is proposed based on current knowledge of nisin's mode of action.

The gene encoding for penicillin-binding protein 5 of Enterococcus faecalis is useful for development of a species-specific DNA probe.

Recently, in Escherichia coli was cloned a Sau3AI 3.4-kb fragment containing the gene encoding for penicillin-binding protein 5 (PBP5) of Enterococcus faecalis. The structural gene for the PBP of E. faecalis and the flanking regions were entirely sequenced (C. Signoretto, M. Boaretti, and P. Canepari, FEMS Microbiol. Lett. 123:99-106, 1994). When the entire cloned E. faecalis DNA insert, labeled with digoxigenin, was used as a probe to detect a homology gene in enterococci, it was observed that only DNAs of all the E. faecalis strains reacted to the probe. The same results were obtained when a HindIII fragment of 0.35 kb from the entire insert of 3.4 kb was used. In this study we tested a total of 62 clinically isolated enterococcal strains, belonging to the species E. faecalis (36 strains), E. faecium (13), E. gallinarum (6), E. bovis (2) E. avium (3), E. hirae (1), and E. casseliflavus (1). The results indicate that both the entire segment and the HindIII fragment may be useful for preparing a species-specific probe for rapid identification of E. faecalis species.

Overproduction of penicillin-binding protein 7 suppresses thermosensitive growth defect at low osmolarity due to an spr mutation of Escherichia coli.

Escherichia coli delta prc mutants lacking periplasmic protease Prc, which was originally found involved in the C-terminal processing of penicillin-binding protein (PBP) 3, show thermosensitive growth at low osmolarity. We isolated thermoresistant revertants containing extragenic suppressor (spr) mutations. In the prc+ background the mutations also caused thermosensitivity at low osmolarity. They were all mapped at about 48 min on the chromosome and most probably allelic to one another. From this chromosomal region we cloned a gene that could correct the thermosensitive defect of an spr mutant, which turned out to be a multicopy suppressor of spr. Analysis of the nucleotide sequence predicted that the gene would code for a low-molecular-weight PBP, and penicillin-binding experiments revealed the product to be PBP 7. Disruption of the gene on the chromosome caused no apparent growth defect. PBP 7 seemed to be degraded by protease Prc. Overproduction of mutant PBP 7 that had the active site serine residue replaced with alanine did not correct the spr thermosensitivity, suggesting importance of the DD-endopeptidase activity in the multicopy suppression.

Cloning, sequencing and expression in Escherichia coli of the low-affinity penicillin binding protein of Enterococcus faecalis.

Low-affinity penicillin binding proteins are particular membrane proteins, in several Gram-positive bacteria, which are involved in beta-lactam antibiotic resistance. The structural gene for the low-affinity penicillin binding protein 5 (PBP5) of Enterococcus faecalis was cloned and sequenced. From the sequence of the 3378 bp, a 2040 bp coding region was identified. From biochemical analysis it emerges that E. faecalis PBP5 is a type II membrane protein with an uncleaved N-terminal and is composed of 679 amino acids with a molecular weight of 74055. This protein showed 48 and 33% of identity with Enterococcus hirae PBP5 and Staphylococcus aureus PBP2a, both low-affinity PBPs involved in beta-lactam resistance. Anti-PBP5 antibodies cross-reacted with a membrane protein present in other species of enterococci, but the entire gene fragment cloned hybridized only with DNAs of E. faecalis strains, thus suggesting that genes coding for low-affinity PBPs of enterococci are not strictly homologous. In this experiment digoxigenin-labelled E. faecalis DNA was used.

Cloning, nucleotide sequence and amplified expression of the gene encoding the extracellular metallo (Zn) DD-peptidase of Streptomyces albus G.

The gene encoding the extracellular metallo (Zn) DD-peptidase of Streptomyces albus G has been cloned in Escherichia coli DH5 alpha MCR via pBR322 or 325, and then transferred into Streptomyces lividans TK24 via pIJ486, with substantial amplification of the expressed DD-peptidase. The gene has the information for the synthesis of a 255 amino acid precursor, the amino terminal region of which has the characteristic features of a signal peptide. The primary structure as deduced from nucleotide sequencing confirms that previously determined by chemical methods except for the occurrence of an Asp instead of Asn at position 1 and an additional Ala immediately downstream of Pro67.

Potentiation of methicillin activity against methicillin-resistant Staphylococcus aureus by diterpenes.

Totarol is a diterpene compound extracted from the totara tree. Totarol and eight other diterpenes were found to potentiate methicillin, one reducing the minimum inhibitory concentration of methicillin against resistant Staphylococcus aureus 256-fold. Totarol did not inhibit the synthesis of DNA or peptidoglycan in S. aureus, but reduced the respiration rate by 70%. Under potentiation conditions, diterpenes had only a slight effect on the respiration rate, but had a significant effect on expression of PBP 2a. We conclude that the primary staphylococcal target for totarol is the respiratory chain, but that potentiation of methicillin by diterpenes is by interference with PBP 2a expression.

In vivo crystal formation in Escherichia coli of an over-expressed soluble form of penicillin-binding protein 5.

Accumulation of either native membrane-bound or soluble variants of PBP5 over-expressed in the cytoplasm was investigated by electron microscopy of ultra-thin sections. One of the soluble forms of PBP5 (PBP5s353) formed well-ordered crystals inside the cells. Cells sectioned perpendicular to their long axis showed a diamond-shaped crystal whereas cells cut parallel to their long axis contained a long, narrow crystal. In both sectioning directions an ordered ultrastructure was visible as shown by optical diffraction. Computer processing was used to enhance the crystal images. From this the unit cell parameters were calculated as a = 7.6 nm, b = 4 nm, c = 4.2 nm, gamma = 75 degrees. The calculated unit-cell volume of 120 nm3 is large enough to contain one protein molecule.

Methicillin resistance in Staphylococcus aureus: mechanisms and modulation.

Staphylococcus aureus is a major pathogen both within hospitals and in the community. Methicillin, a beta-lactam antibiotic, acts by inhibiting penicillin-binding proteins (PBPs) that are involved in the synthesis of peptidoglycan, an essential mesh-like polymer that surrounds the cell. S. aureus can become resistant to methicillin and other beta-lactam antibiotics through the expression of a foreign PBP, PBP2a, that is resistant to the action of methicillin but which can perform the functions of the host PBPs. Methicillin-resistant S. aureus isolates are often resistant to other classes of antibiotics (through different mechanisms) making treatment options limited, and this has led to the search for new compounds active against these strains. An understanding of the mechanism of methicillin resistance has led to the discovery of accessory factors that influence the level and nature of methicillin resistance. Accessory factors, such as Fem factors, provide possible new targets, while compounds that modulate methicillin resistance such as epicatechin gallate, derived from green tea, and corilagin, provide possible lead compounds for development of inhibitors.

Synergy of sulbactam and ampicillin against methicillin-resistant staphylococci.

Methicillin resistance in staphylococci is an increasing problem both for Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), which cause infections of the heart and after central nervous system surgery. Resistance seems to be due primarily to production of altered penicillin-binding proteins. The present study determined whether a combination of beta-lactamase inhibitor sulbactam and ampicillin or sulbactam and cefazolin would inhibit MRSA and MRSE. Sulbactam, ampicillin and cefazolin at 32 micrograms/ml did not inhibit MRSA or MRSE. At 8 micrograms/ml of each agent all isolates were inhibited. Synergy of sulbactam and ampicillin could be demonstrated against MRSA by the agar fixed ratio method, checkerboard dilution and by killing curves. This suggests that in certain situations MRSA and MRSE may be effectively eliminated by this method.

[Detection of methicillin-resistant Staphylococcus aureus by polymerase chain reaction].

The low-affinity penicillin-binding protein (PBP 2') is associated with methicillin-resistance of Staphylococcus aureus and its structural gene (mecA) not present in methicillin-susceptible S. aureus could be detected by the polymerase chain reaction (PCR) method, in which a 533 bp region of mecA was amplified and detected by agarose gel electrophoresis. Survey for the mecA gene in 210 clinical isolates of S. aureus revealed that, while there was a gross correlation between the presence of the mecA gene and the resistance level to beta-lactams, three strains of mecA (+) tested showed beta-lactam susceptibility similar to those of mecA (-) strains. These three strains did not produce a detectable amount of PBP 2' constitutively nor inducibly, which was the cause of their high susceptibility to beta-lactams. One of them yielded a typical methicillin-resistant variant at a low frequency with a concomitant recovery of PBP 2' production when the bacterial cells of high density were spread onto an agar plate containing 10 micrograms/ml of oxacillin. These findings suggested that typical methicillin resistant S. aureus occurred during chemotherapy with beta-lactam antibiotics even when resistant strains could not be detected by the susceptibility test and thus all mecA (+) strains including those with high susceptibility should be precisely detected.

A detailed study of gerJ mutants of Bacillus subtilis.

A total of nine gerJ mutants have now been isolated in Bacillus subtilis. All are defective in their spore germination properties, being blocked at an intermediate (phase grey) stage. The dormant spores are sensitive to heating at 90 degrees C and two of the mutants (generated by transposon insertion) produce spores sensitive at 80 degrees C. The spores of these two more extreme mutants had a visibly defective cortex when studied by electron microscopy, as did some of the other mutants. During sporulation, the acquisition of spore resistance properties and the appearance of the sporulation-specific penicillin-binding protein PBP5* were delayed. A strain probably carrying a lacZ fusion to the gerJ promoter demonstrated increased expression between t2 and t4. We propose that the gerJ locus is involved in the control of one or more sporulation-specific genes.