Activity of the two-component regulatory system CiaRH in Streptococcus pneumoniae R6

The two-component regulatory system CiaRH of Streptococcus pneumoniae affects a variety of processes such as competence development, autolysis, bacteriocin production, host colonization, and virulence. While the targets of the regulator CiaR are known, the role of phosphorylation in CiaR regulation has not been defined. To address this issue, the presumed phosphorylation site of CiaR, aspartic acid at position 51, was replaced by alanine. The mutant CiaRD51A protein was no longer able to activate CiaR-dependent promoters, strongly suggesting that the phosphorylated form of CiaR is active in regulation. However, depending on the growth medium, inactivation of the kinase gene ciaH resulted in a subtle increase of CiaR-dependent promoter activities or in a strong reduction. Therefore, CiaH may act as a kinase or phosphatase and CiaR is apparently able to obtain its phosphate independently of CiaH. On the other hand, promoter measurements in cells with an intact CiaRH system demonstrated a high, nearly constitutive, expression level of the CiaR regulon independent from the growth medium. Thus, in contrast to many other two-component regulatory systems, CiaRH has apparently evolved to maintain high levels of gene expression under a variety of conditions rather than responding strongly to a signal.     

A two-component signal-transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae

Penicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin-binding protein (PBP) variants with decreased affinities for β-lactam antibiotics. Cefotaxime-resistant laboratory mutants, selected after several steps on increasing concentrations of this β-lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, claR and claH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal-transducing proteins). Two hydrophobic peptide regions divided the histidine kinase ClaH into two putative domains: an N-terminal extracelluiar sensor part, and an intracelluiar C-terminal domain with the conserved His-226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime-resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime-resistant mutants were each located in the C-terminal half of ClaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated claH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal-transducing system cia is involved in early steps of competence regulation.     

β-Lactam resistance in Streptococcus pneumoniae: penicillin-binding proteins and non-penicillin-binding proteins

The beta-lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of beta-lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no beta-lactamase-producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the beta-lactams, the penicillin-binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non-PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non-PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non-PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during beta-lactam treatment counteract the effects caused by the inhibition of penicillin-binding proteins.     

The CiaRH system of Streptococcus pneumoniae prevents lysis during stress induced by treatment with cell wall inhibitors and by mutations in pbp2x involved in beta-lactam resistance

The two-component signal-transducing system CiaRH of Streptococcus pneumoniae plays an important role during the development of beta-lactam resistance in laboratory mutants. We show here that a functional CiaRH system is required for survival under many different lysis-inducing conditions. Mutants with an activated CiaRH system were highly resistant to lysis induced by a wide variety of early and late cell wall inhibitors, such as cycloserine, bacitracin, and vancomycin, and were also less susceptible to these drugs. In contrast, loss-of-function CiaRH mutants were hypersusceptible to these drugs and were apparently unable to maintain a stationary growth phase in normal growth medium and under choline deprivation as well. Moreover, disruption of CiaR in penicillin-resistant mutants with an altered pbp2x gene encoding low-affinity PBP2x resulted in severe growth defects and rapid lysis. This phenotype was observed with pbp2x genes containing point mutations selected in the laboratory and with highly altered mosaic pbp2x genes from penicillin-resistant clinical isolates as well. This documents for the first time that PBP2x mutations required for development of beta-lactam resistance are functionally not neutral and are tolerated only in the presence of the CiaRH system. This might explain why cia mutations have not been observed in penicillin-resistant clinical isolates. The results document that the CiaRH system is required for maintenance of the stationary growth phase and for prevention of autolysis triggered under many different conditions, suggesting a major role for this system in ensuring cell wall integrity.     

Pneumococcal HtrA protease mediates inhibition of competence by the CiaRH two-component signaling system

Activation of the CiaRH two-component signaling system prevents the development of competence for genetic transformation in Streptococcus pneumoniae through a previously unknown mechanism. Earlier studies have shown that CiaRH controls the expression of htrA, which we show encodes a surface-expressed serine protease. We found that mutagenesis of the putative catalytic serine of HtrA, while not impacting the competence of a ciaRH+ strain, restored a normal competence profile to a strain having a mutation that constitutively activates the CiaH histidine kinase. This result implies that activity of HtrA is necessary for the CiaRH system to inhibit competence. Consistent with this finding, recombinant HtrA (rHtrA) decreased the competence of pneumococcal cultures. The rHtrA-mediated decline in transformation efficiency could not be corrected with excess competence-stimulating peptide (CSP), suggesting that HtrA does not act through degradation of this signaling molecule. The inhibitory effects of rHtrA and activated CiaH, however, were largely overcome in a strain having constitutive activation of the competence pathway through a mutation in the cytoplasmic domain of the ComD histidine kinase. Although these results suggested that HtrA might act through degradation of the extracellular portion of the ComD receptor, Western immunoblots for ComD did not reveal changes in protein levels attributable to HtrA. We therefore postulate that HtrA may act on an unknown protein target that potentiates the activation of the ComDE system by CSP. These findings suggest a novel regulatory role for pneumococcal HtrA in modulating the activity of a two-component signaling system that controls the development of genetic competence.     

Diversity of penicillin-binding proteins. Resistance factor FmtA of Staphylococcus aureus

Antibiotic-resistant Staphylococcus aureus is a major concern to public health. Methicillin-resistant S. aureus strains are completely resistant to all beta-lactams antibiotics. One of the main factors involved in methicillin resistance in S. aureus is the penicillin-binding protein, PBP2a. This protein is insensitive to inactivation by beta-lactam antibiotics such as methicillin. Although other proteins are implicated in high and homogeneous levels of methicillin resistance, the functions of these other proteins remain elusive. Herein, we report for the first time on the putative function of one of these proteins, FmtA. This protein specifically interacts with beta-lactam antibiotics forming covalently bound complexes. The serine residue present in the sequence motif Ser-X-X-Lys (which is conserved among penicillin-binding proteins and beta-lactamases) is the active-site nucleophile during the formation of acyl-enzyme species. FmtA has a low binding affinity for beta-lactams, and it experiences a slow acylation rate, suggesting that this protein is intrinsically resistant to beta-lactam inactivation. We found that FmtA undergoes conformational changes in presence of beta-lactams that may be essential to the beta-lactam resistance mechanism. FmtA binds to peptidoglycan in vitro. Our findings suggest that FmtA is a penicillin-binding protein, and as such, it may compensate for suppressed peptidoglycan biosynthesis under beta-lactam induced cell wall stress conditions.     

Bora Downregulation Results in Radioresistance by Promoting Repair of Double Strand Breaks

Following DNA double-strand breaks cells activate several DNA-damage response protein kinases, which then trigger histone H2AX phosphorylation and the accumulation of proteins such as MDC1, p53-binding protein 1, and breast cancer gene 1 at the damage site to promote DNA double-strand breaks repair. We identified a novel biomarker, Bora (previously called C13orf34), that is associated with radiosensitivity. In the current study, we set out to investigate how Bora might be involved in response to irradiation. We found a novel function of Bora in DNA damage repair response. Bora down-regulation increased colony formation in cells exposed to irradiation. This increased resistance to irradiation in Bora-deficient cells is likely due to a faster rate of double-strand breaks repair. After irradiation, Bora-knockdown cells displayed increased G2-M cell cycle arrest and increased Chk2 phosphorylation. Furthermore, Bora specifically interacted with the tandem breast cancer gene 1 C-terminal domain of MDC1 in a phosphorylation dependent manner, and overexpression of Bora could abolish irradiation induced MDC1 foci formation. In summary, Bora may play a significant role in radiosensitivity through the regulation of MDC1 and DNA repair.     

Inducible degradation of checkpoint kinase 2 links to cisplatin-induced resistance in ovarian cancer cells

Checkpoint kinase 2 (Chk2) is one of the critical kinases governing the cell cycle checkpoint, DNA damage repair, and cell apoptosis in response to DNA damaging signals. In the present report, we demonstrate that Chk2 kinase is degraded at the protein level in response to cisplatin through ubiquitin-proteasome pathway. This degradation was independent of the Thr68 phosphorylation, ATM kinase, and BRCA1 tumor suppressor. Examination of Chk2 protein revealed a decreased expression of Chk2 protein in cisplatin-resistant ovarian cancer cell lines, suggesting that degradation or decreased expression of Chk2 is partially responsible for chemo-resistance. Site-directed mutation of the putative destruction box in the Chk2 protein did not affect the Chk2 degradation induced by cisplatin. Therefore, these results are the first to indicate a novel mechanism of regulating Chk2 in cisplatin-induced resistance of cancer cells.     

Hyperactivation of the yeast DNA damage checkpoint by TEL1 and DDC2 overexpression.

The evolutionarily conserved yeast Mec1 and Tel1 protein kinases, as well as the Mec1-interacting protein Ddc2, are involved in the DNA damage checkpoint response. We show that regulation of Tel1 and Ddc2-Mec1 activities is important to modulate both activation and termination of checkpoint-mediated cell cycle arrest. In fact, overproduction of either Tel1 or Ddc2 causes a prolonged cell cycle arrest and cell death in response to DNA damage, impairing the ability of cells to recover from checkpoint activation. This cell cycle arrest is independent of Mec1 in UV-irradiated Tel1-overproducing cells, while it is strictly Mec1 dependent in similarly treated DDC2-overexpressing cells. The Rad53 checkpoint kinase is instead required in both cases for cell cycle arrest, which correlates with its enhanced and persistent phosphorylation, suggesting that unscheduled Rad53 phosphorylation might prevent cells from re-entering the cell cycle after checkpoint activation. In addition, Tel1 overproduction results in transient nuclear division arrest and concomitant Rad53 phosphorylation in the absence of exogenous DNA damage independently of Mec1 and Ddc1.     

A Functional Link Between SIRT1 Deacetylase and NBS1 in DNA Damage Response

In mammalian cells, DNA is often subjected to stresses such as ionizing radiation (IR) and ultraviolet light that can induce DNA double strand breaks (DSBs). In response to DNA DSBs, mammalian cells activate a rapid phosphorylation signaling cascade through the protein kinases, Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-Related (ATR).1 Many well-characterized DNA repair factors are phosphorylated by ATM in response to DSBs, and the sequential phosphorylation of some of these factors, including NBS1, delay cell cycle progression (checkpoint arrest) to allow time for DNA damage repair.2 Results from a new study suggest that phosphorylation of NBS1 is regulated by the acetylation status of the protein, which is modulated by SIRT1 deacetylase.     

Lysis of Escherichia coli by beta-lactam antibiotics: deletion analysis of the role of penicillin-binding proteins 1A and 1B

Deletions of the ponA and ponB genes of Escherichia coli have been constructed in vitro and recombined into the chromosome to produce strains that completely lack penicillin-binding protein 1A or penicillin-binding protein 1B. In each case a DNA fragment internal to the gene was replaced by a fragment encoding an antibiotic resistance. The ponA and ponB deletions can therefore be readily introduced into other E. coli strains by P1 transduction of the antibiotic resistance. Although the complete absence of penicillin-binding protein 1A or penicillin-binding protein 1B was tolerated, the absence of both of these proteins was shown to result in bacterial lysis.     

Solving staphylococcal resistance to beta-lactams

Class resistance to beta-lactam antibiotics in Gram-positive bacteria is mediated by structural changes in transpeptidase penicillin-binding proteins. These structural changes render a complex series of interactions between antibiotic and protein that are energetically unfavorable, such that the active site is inactivated not at all or too slowly to prevent cell-wall synthesis and bacterial growth. Determination of the crystal structure of the low-affinity penicillin-binding protein PBP2a, which mediates beta-lactam antibiotic resistance in staphylococci, has identified the molecular structures and interactions that are responsible for resistance. This information could be useful for designing beta-lactams to overcome these structural impediments, as well as resistance.