Cell Wall-active Antibiotics Research Articles

Cell wall stress responses in Bacillus subtilis: the regulatory network of the bacitracin stimulon.

In response to sublethal concentrations of antibiotics, bacteria often induce an adaptive response that can contribute to antibiotic resistance. We report the response of Bacillus subtilis to bacitracin, an inhibitor of cell wall biosynthesis found in its natural environment. Analysis of the global transcriptional profile of bacitracin-treated cells reveals a response orchestrated by two alternative sigma factors (sigmaB and sigmaM) and three two-component systems (YvqEC, YvcPQ and BceRS). All three two-component systems are located next to target genes that are strongly induced by bacitracin, and the corresponding histidine kinases share an unusual topology: they lack about 100 amino acids in their extracellular sensing domain, which is almost entirely buried in the cytoplasmic membrane. Sequence analysis indicates that this novel N-terminal sensing domain is a characteristic feature of a subfamily of histidine kinases, found almost entirely in Gram-positive bacteria and frequently linked to ABC transporters. A systematic mutational analysis of bacitracin-induced genes led to the identification of a new bacitracin-resistance determinant, bceAB, encoding a putative ABC transporter. The bcrC bacitracin resistance gene, which is under the dual control of sigmaX and sigmaM, was also induced by bacitracin. By comparing the bacitracin and the vancomycin stimulons, we can differentiate between loci induced specifically by bacitracin and those that are induced by multiple cell wall-active antibiotics.

CesRK, a two-component signal transduction system in Listeria monocytogenes, responds to the presence of cell wall-acting antibiotics and affects beta-lactam resistance.

Listeria monocytogenes is a food-borne pathogen that can cause a variety of illnesses ranging from gastroenteritis to life-threatening septicemia. The beta-lactam antibiotic ampicillin remains the drug of choice for the treatment of listeriosis. We have previously identified a response regulator of a putative two-component signal transduction system that plays a role in the virulence and ethanol tolerance of L. monocytogenes. Here we present evidence that the response regulator, CesR, and a histidine protein kinase, CesK, which is encoded by the gene downstream from cesR, are involved in the ability of L. monocytogenes to tolerate ethanol and cell wall-acting antibiotics of the beta-lactam family. Furthermore, CesRK controls the expression of a putative extracellular peptide encoded by the orf2420 gene, located immediately downstream from cesRK. Inactivation of orf2420 revealed that it contributes to ethanol tolerance and pathogenesis in mice. Interestingly, we found that transcription of orf2420 was strongly induced by subinhibitory concentrations of various cell wall-acting antibiotics, ethanol, and lysozyme. The induction of orf2420 expression was abolished in the absence of CesRK. Our data suggest that CesRK is involved in regulating aspects of the cell envelope architecture and that changes in cell wall integrity provide a potent stimulus for CesRK-mediated regulation. These results further our understanding of how L. monocytogenes senses and responds to antibiotics that are used therapeutically in the treatment of infectious diseases.

Transcriptional Induction of the Penicillin-Binding Protein 2 Gene in Staphylococcus aureus by Cell Wall-Active Antibiotics Oxacillin and Vancomycin

Transcriptional Induction of the Penicillin-Binding Protein 2 Gene in <i>Staphylococcus aureus</i> by Cell Wall-Active Antibiotics Oxacillin and Vancomycin

Transcriptional induction of the penicillin-binding protein 2 gene in Staphylococcus aureus by cell wall-active antibiotics oxacillin and vancomycin.

We found an increased abundance of pbpB-specific transcripts in vancomycin intermediate-resistant Staphylococcus aureus (VISA) isolates compared with that found in paired, genetically identical, susceptible isolates. This difference in expression cannot be explained by differences in the pbpB promoter sequence. Since the factors controlling pbpB gene expression have remained largely unexplored, various conditions that might affect pbpB transcript abundance were examined. In both vancomycin-susceptible and VISA strains, pbpB expression varied with the growth phase, with the highest abundance of pbpB-specific transcripts detected during mid-log phase. Interestingly, both vancomycin and oxacillin were able to induce pbpB transcription above a constitutive level. When vancomycin was absent, one of the three pbpB-specific transcripts that were usually faintly detected in non-VISA strains was more readily detected in VISA strains during mid-log but not stationary phase. This transcript was enhanced in non-VISA strains by vancomycin induction. Gel shift assays indicated that an increased amount of the putative transcription factor that binds to both P1 and P1' promoter regions is present in the cytosol of vancomycin-induced cells. Neither the SigB sigma factor nor the quorum-sensing agr locus was required for growth phase-variable pbpB expression or transcriptional induction of pbpB by vancomycin or oxacillin. Also, MecI, MecR1, BlaI, and BlaR1, regulatory proteins that mediate beta-lactam-inducible expression of mecA and beta-lactamase, were not required for antibiotic induction of pbpB transcription. These data support the idea that pbpB expression is modulated by a trans-acting factor in response to the presence of the cell wall-active antibiotics vancomycin and oxacillin.

Impact of sigB mutation on Staphylococcus aureus oxacillin and vancomycin resistance varies with parental background and method of assessment

Previous studies of Staphylococcus aureus transposon insertion mutants showing decreased methicillin or teicoplanin resistance have suggested a role for the RNA polymerase alternative sigma factor SigB in the expression of resistance to these antibiotics. A knockout mutation was created in the S. aureus strain COL sigB gene and its influence on oxacillin and vancomycin resistance was studied in a variety of parental backgrounds. Typically, sigB mutants of methicillin-resistant strains had oxacillin minimum inhibitory concentrations (MICs) one-half of their parent strains. The effect of the sigB mutation appeared to be more dramatic when assessed by population analysis profiles or by growth in liquid culture in shaking flasks than by MIC determinations. Oxacillin MICs of COL and the COLΔ sigB mutant were 400 and 200 mg/l, respectively, by conventional determination and 800 and 100–200 mg/l from population analysis profiles. The COLΔ sigB mutant strain was significantly more inhibited by a range of oxacillin concentrations in a shake flask culture than strain COL. Mutation of sigB caused a decrease in vancomycin resistance in two laboratory derived glycopeptide-intermediate S. aureus strains. The results suggest that some protein products whose expression is controlled by SigB play a role in resistance to cell wall-active antibiotics.

In vitro and in vivo functional activity of Chlamydia MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance.

Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal DeltamurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.

Characterization of the depletion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase in Escherichia coli and Bacillus subtilis.

The ispF gene product in Escherichia coli has been shown to catalyze the formation of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC) in the deoxyxylulose (DOXP) pathway for isoprenoid biosynthesis. In this work, the E. coli gene ispF and its Bacillus subtilis orthologue, yacN, were deleted and conditionally complemented by expression of these genes from distant loci in the respective organisms. In E. coli, complementation was achieved through integration of ispF at the araBAD locus with control from the arabinose-inducible araBAD promoter, while in B. subtilis, yacN was placed at amyE under control of the xylose-inducible xylA promoter. In both cases, growth was severely retarded in the absence of inducer, consistent with these genes being essential for survival. E. coli cells depleted of MEC synthase revealed a filamentous phenotype. This was in contrast to the depletion of MEC synthase in B. subtilis, which resulted in a loss of rod shape, irregular septation, multicompartmentalized cells, and thickened cell walls. To probe the nature of the predominant deficiency of MEC synthase-depleted cells, we investigated the sensitivity of these conditionally complemented mutants, grown with various concentrations of inducer, to a wide variety antibiotics. Synthetic lethal behavior in MEC synthase-depleted cells was prevalent for cell wall-active antibiotics.

Chemistry and biology of the ramoplanin family of peptide antibiotics.

The peptide antibiotic ramoplanin factor A2 is a promising clinical candidate for treatment of Gram-positive bacterial infections that are resistant to antibiotics such as glycopeptides, macrolides, and penicillins. Since its discovery in 1984, no clinical or laboratory-generated resistance to this antibiotic has been reported. The mechanism of action of ramoplanin involves sequestration of peptidoglycan biosynthesis Lipid intermediates, thus physically occluding these substrates from proper utilization by the late-stage peptidoglycan biosynthesis enzymes MurG and the transglycosylases (TGases). Ramoplanin is structurally related to two cell wall active lipodepsipeptide antibiotics, janiemycin, and enduracidin, and is functionally related to members of the lantibiotic class of antimicrobial peptides (mersacidin, actagardine, nisin, and epidermin) and glycopeptide antibiotics (vancomycin and teicoplanin). Peptidomimetic chemotherapeutics derived from the ramoplanin sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and related pathogens. Here we review the chemistry and biology of the ramoplanins including its discovery, structure elucidation, biosynthesis, antimicrobial activity, mechanism of action, and total synthesis.

Enterococcal endocarditis in Sweden, 1995-1999: can shorter therapy with aminoglycosides be used?

A 5-year nationwide prospective study in Sweden during 1995-1999 identified 881 definite episodes of infective endocarditis. Definite enterococcal endocarditis was diagnosed in 93 episodes (11%), the largest series of enterococcal endocarditis so far presented. Mortality during treatment was 16%, the relapse rate was 3%, and clinical cure was achieved in the remaining 81% of the episodes. Clinical cure was achieved with a median duration of cell wall-active antimicrobial therapy of 42 days combined with an aminoglycoside (median treatment time, 15 days). International guidelines generally recommend a 4-6-week combined synergistic treatment course with a cell wall-active antibiotic and an aminoglycoside. Treatment regimens in Sweden often include a shortened aminoglycoside treatment course in order to minimize adverse effects in older patients. Fatal outcome seemed not to be due to the shortened aminoglycoside therapy course. In many enterococcal endocarditis episodes, duration of aminoglycoside therapy could probably be shortened to 2-3 weeks.

The role of murMN operon in penicillin resistance and antibiotic tolerance of Streptococcus pneumoniae.

The recently identified murMN operon is essential for the production of branched-structured muropeptides in the cell wall and also for the expression of the resistant phenotype in penicillin-resistant strains of Streptococcus pneumoniae. The purpose of studies described in this communication was to understand better the role of murMN in penicillin resistance. Deletion of murM in the penicillin-resistant strain Pen6, which causes reduction in the penicillin MIC from 6.0 to 0.03 microg/ml, was successfully complemented to recover the original high level of penicillin resistance in transformants that received functional murM alleles cloned in plasmid pLS578. Inactivation of penicillin resistance was not accompanied by any detectable change in the low affinity or abnormal molecular size pattern of the penicillin-binding proteins (PBPs) nor in the mosaic sequence of PBP2X typical of resistant strain Pen6. Exposure of strain Pen6 with inactivated murM to 0.05 microg/ml of penicillin (i.e., a concentration more than 100 times below the MIC of the parental strain) initiated a phenotypic response typical of penicillin-susceptible strains of pneumococci: inhibition of growth followed by rapid and extensive loss of viability and lysis. Unexpectedly, inactivation of murMN also caused hypersensitivity to lysis by low concentrations of a variety of cell wall active antibiotics such as fosfomycin, D-cycloserine, and nisin, suggesting that the murMN operon may perform an important regulatory role in the control of the irreversible antimicrobial effects of cell wall inhibitors.

Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach

Proteins produced in elevated amounts in response to oxacillin challenge of Staphylococcus aureus strain RN450, were studied by comparing Coomassie blue stained two-dimensional gels of cellular proteins. At least nine proteins were produced in elevated amounts following exposure to growth inhibitory concentrations of oxacillin. N-terminal sequences were obtained for five of the proteins and the databases were searched to tentatively identify them. The proteins were identified as homologs of (i) methionine sulfoxide reductase (MsrA); (ii) a signal transduction protein (TRAP) involved in regulating RNAIII production encoded by the agr locus; (iii) transcription elongation factor GreA; (iv) the heat shock protein GroES; and (v) the enzyme IIA component of the phosphoenolpyruvate:sugar phosphotransferase system. A similar induction response was observed with the other cell wall-active antibiotics, but not with antibiotics that affect other cellular targets. Increased transcription of the msrA and groEL genes in response to cell wall-active antibiotics was also demonstrated. Although net protein synthesis is inhibited subsequent to inhibition of peptidoglycan biosynthesis by cell wall-active antibiotics, some proteins are induced in S. aureus, presumably in an attempt by the cell to counter the inhibitory effects of these agents.

Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach.

Proteins produced in elevated amounts in response to oxacillin challenge of Staphylococcus aureus strain RN450, were studied by comparing Coomassie blue stained two-dimensional gels of cellular proteins. At least nine proteins were produced in elevated amounts following exposure to growth inhibitory concentrations of oxacillin. N-terminal sequences were obtained for five of the proteins and the databases were searched to tentatively identify them. The proteins were identified as homologs of (i) methionine sulfoxide reductase (MsrA); (ii) a signal transduction protein (TRAP) involved in regulating RNAIII production encoded by the agr locus; (iii) transcription elongation factor GreA; (iv) the heat shock protein GroES; and (v) the enzyme IIA component of the phosphoenolpyruvate:sugar phosphotransferase system. A similar induction response was observed with the other cell wall-active antibiotics, but not with antibiotics that affect other cellular targets. Increased transcription of the msrA and groEL genes in response to cell wall-active antibiotics was also demonstrated. Although net protein synthesis is inhibited subsequent to inhibition of peptidoglycan biosynthesis by cell wall-active antibiotics, some proteins are induced in S. aureus, presumably in an attempt by the cell to counter the inhibitory effects of these agents.

Effects of antibiotic class on the macrophage inflammatory response to Streptococcus pneumoniae.

Antibiotic choice can alter host inflammation during invasive bacterial infections. Previous studies of gram-negative organisms concluded that antibiotic-mediated release of bacterial cell wall components amplifies inflammation. Less has been reported about antibiotic effect on gram-positive organisms. This study explored the hypothesis that Streptococcus pneumoniae would induce greater macrophage inflammatory mediator production when killed with cell wall active antibiotics rather than protein synthesis inhibitors. Stimulation of RAW 264.7 murine macrophages with pneumococci and oxacillin led to significantly higher inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation than did the same concentrations of pneumococci and clindamycin. Neither antibiotic alone or in combination with lipopolysaccharide acted directly on macrophages to modify the immune response. Endotoxin contamination did not confound the results, as preincubation with polymyxin B did not change iNOS or TNF protein levels. Thus, the antimicrobial mechanism of action affects macrophage inflammatory mediator production after stimulation with pneumococci.

Increasing Antimicrobial Resistance: Therapeutic Implications for Enterococcal Infections.

Enterococcus was designated a genus distinct from the streptococci in 1984. Enterococci cause a variety of monomicrobial and polymicrobial infections, mainly in compromised patients. These infections include bacteremia, urinary and biliary tract infections, intra-abdominal sepsis, and decubitus and diabetic foot ulcers. Enterococcal infections may be acquired from the patient's endogenous intestinal flora or exogenously from a fecally contaminated environment. Enterococci are inherently resistant to many antimicrobial agents and readily acquire additional resistances, which is likely the reason that enterococci have become prominent nosocomial pathogens. Only the combination of a cell wall-active antibiotic to which the Enterococcus is susceptible (ie, certain beta-lactams or vancomycin) plus an aminoglycoside (ie, gentamicin or streptomycin) is bactericidal, and is required for cure of endocarditis, meningitis and probably infection in neutropenic patients; bacteriostatic activity is sufficient to treat most other infections. Treatment of infections caused by strains resistant to beta-lactams, glycopeptides and aminoglycosides has become problematic due the limited number of therapeutic options. No medical therapy is reliably effective for endocarditis caused by strains resistant to all cell wall-active antibiotics and all aminoglycosides. New antimicrobial agents, such as linezolid and quinupristin/dalfopristin, have recently become available, but their activity against enterococci is mainly bacterostatic.

Characterization of the Mycobacterium tuberculosis iniBAC promoter, a promoter that responds to cell wall biosynthesis inhibition.

The cell wall provides an attractive target for antibiotics against Mycobacterium tuberculosis. Agents such as isoniazid and ethambutol that work by inhibiting cell wall biosynthesis are among the most highly effective antibiotics against this pathogen. Although considerable progress has been made identifying the targets for cell wall active antibiotics, little is known about the intracellular mechanisms that are activated as a consequence of cell wall injury. These mechanisms are likely to have an important role in growth regulation and in the induction of cell death by antibiotics. We previously discovered three isoniazid-induced genes (iniB, iniA, and iniC) organized in tandem on the M. tuberculosis genome. Here, we investigate the unique features of the putative iniBAC promoter. This promoter was specifically induced by a broad range of inhibitors of cell wall biosynthesis but was not inducible by other conditions that are toxic to mycobacteria via other mechanisms. Induction required inhibitory concentrations of antibiotics and could be detected only in actively growing cells. Analysis of the iniBAC promoter sequence revealed both a regulatory element upstream and a potential repressor binding region downstream of the transcriptional start site. The induction phenotype and structure of the iniBAC promoter suggest that a complex intracellular response occurs when cell wall biosynthesis is inhibited in M. tuberculosis and other mycobacteria.

Antifungal metabolites (monorden, monocillin IV, and cerebrosides) from Humicola fuscoatra traaen NRRL 22980, a mycoparasite of Aspergillus flavus sclerotia.

The mycoparasite Humicola fuscoatra NRRL 22980 was isolated from a sclerotium of Aspergillus flavus that had been buried in a cornfield near Tifton, Ga. When grown on autoclaved rice, this fungus produced the antifungal metabolites monorden, monocillin IV, and a new monorden analog. Each metabolite produced a clear zone of inhibition surrounding paper assay disks on agar plates seeded with conidia of A. flavus. Monorden was twice as inhibitory to A. flavus mycelium extension (MIC > 28 microg/ml) as monocillin IV (MIC > 56 microg/ml). Cerebrosides C and D, metabolites known to potentiate the activity of cell wall-active antibiotics, were separated from the ethyl acetate extract but were not inhibitory to A. flavus when tested as pure compounds. This is the first report of natural products from H. fuscoatra.

Tumor necrosis factor alpha and interleukin 6 release induced by antibiotic killing of Pseudomonas aeruginosa and Staphylococcus aureus.

Using a recently described ex vivo model, tumor necrosis factor-alpha and interleukin-6 released by peripheral blood monocytes after killing of Pseudomonas aeruginosa and Staphylococcus aureus by ceftazidime, imipenem, and meropenem was measured. Cytokine release was highest with ceftazidime and lowest with imipenem for both bacteria (p < 0.05), although cytokine concentrations were much lower after killing of Staphylococcus aureus. Differences in cytokine release rates induced by various cell-wall active antibiotics have not yet been described for gram-positive organisms and should be studied further.

Differential release of smooth-type lipopolysaccharide from Pseudomonas aeruginosa treated with carbapenem antibiotics and its relation to production of tumor necrosis factor alpha and nitric oxide

Endotoxin release from Pseudomonas aeruginosa treated with cell wall-active carbapenem antibiotics and its effect on the production of tumor necrosis factor alpha and nitric oxide were examined. Treatment of bacteria with imipenem induced much lower levels of endotoxin release than treatment with meropenem. The endotoxin released was demonstrated to be of the smooth type and O-specific polysaccharide-rich. The exposure of the filtrates of P. aeruginosa treated with imipenem to physiologically relevant cells caused low-level production of tumor necrosis factor alpha and nitric oxide, while similar treatment with meropenem induced high levels of production.

Cell wall changes in nisin-resistant variants of Listeria innocua grown in the presence of high nisin concentrations

Two nisin-resistant variants of a strain of Listeria innocua were isolated after growth in the presence of 500 and 4000 IU ml −1 of nisin A showed increased cell wall hydrophobicity, resistance to phage attack and three different cell wall-acting antibiotics, as well as to the peptidoglycan hydrolytic enzymes lysozyme and mutanolysin, as compared to the parental strain. Transmission electron microscopy revealed marked thickening of the wall of nisin-resistant cells with an irregular surface. Differences in thickness were lost after cell wall purification and no significant difference in gross wall composition was observed between the parental and resistant variants. Cell wall changes in nisin-resistant listeriae are attributed to abnormal cell wall synthesis and autolysin inhibition, the latter possibly associated with subtle changes in cell wall structures and function.

Contribution of soluble endotoxin released from Gram-negative bacteria by antibiotics to the pathogenesis of experimental sepsis in mice

The results from our laboratory summarized in this review support the importance of endotoxin release from the Gram-negative microbe as a requirement for the full manifestation of the biological activity of the lipopolysaccharide (LPS) macromolecule. They further suggest that LPS is the most important bacterial component released from Gram-negative microbes treated in vitro with cell-wall active antibiotics in terms of biological activity. The use of several experimental mouse models of infection confirm that mice treated with the reversible hepatotoxin, D-galactosamine, are sensitive to both endotoxin lethality and to infection with Gram-negative microbes. Infected LPS-sensitive animals can be protected if they are treated with antibiotics, but the degree of protection (e.g. 5-10-fold) is significantly less than that observed when infection-sensitive but LPS-insensitive mice are similarly treated (500-fold protection). Additional levels of protection in infected, antibiotic-treated mice can be observed with antiendotoxin agents (e.g. polymyxin B-dextran, monoclonal antibody). Finally, antibiotics with equivalent minimal inhibitory concentrations for Escherichia coli, but differing in their mode of action and endotoxin releasing potential in vitro, also differ in their protective efficacy in vivo to reduce mortality in infected animals. Collectively, these data provide evidence to support a role for microbe-derived endotoxin, released from the microbial surface following treatment with cell-wall active antibiotics, in the pathogenesis of experimental Gram-negative sepsis.