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ABSTRACT: A major contributor to the emergence of antibiotic resistance in Gram-positive bacterial pathogens is the expansion of acquired, inducible genetic elements. Although acquired, inducible antibiotic resistance is not new, the interest in its molecular basis has been accelerated by the widening distribution and often 'silent' spread of the elements responsible, the diagnostic challenges of such resistance and the mounting limitations of available agents to treat Gram-positive infections. Acquired, inducible antibiotic resistance elements belong to the accessory genome of a species and are horizontally acquired by transformation/recombination or through the transfer of mobile DNA elements. The two key, but mechanistically very different, induction mechanisms are: ribosome-sensed induction, characteristic of the macrolide-lincosamide-streptogramin B antibiotics and tetracycline resistance, leading to ribosomal modifications or efflux pump activation; and resistance by cell surface-associated sensing of β-lactams (e.g., oxacillin), glycopeptides (e.g., vancomycin) and the polypeptide bacitracin, leading to drug inactivation or resistance due to cell wall alterations.
Future Microbiology 08/2012; 7(8):959-78. · 3.82 Impact Factor
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ABSTRACT: The antimicrobial efflux system encoded by the operon mef(E)-mel on the mobile genetic element MEGA in Streptococcus pneumoniae and other Gram-positive bacteria is inducible by macrolide antibiotics and antimicrobial peptides. Induction may affect the clinical response to the use of macrolides. We developed mef(E) reporter constructs and a disk diffusion induction and resistance assay to determine the kinetics and basis of mef(E)-mel induction. Induction occurred rapidly, with a >15-fold increase in transcription within 1 h of exposure to subinhibitory concentrations of erythromycin. A spectrum of environmental conditions, including competence and nonmacrolide antibiotics with distinct cellular targets, did not induce mef(E). Using 16 different structurally defined macrolides, induction was correlated with the amino sugar attached to C-5 of the macrolide lactone ring, not with the size (e.g., 14-, 15- or 16-member) of the ring or with the presence of the neutral sugar cladinose at C-3. Macrolides with a monosaccharide attached to C-5, known to block exit of the nascent peptide from the ribosome after the incorporation of up to eight amino acids, induced mef(E) expression. Macrolides with a C-5 disaccharide, which extends the macrolide into the ribosomal exit tunnel, disrupting peptidyl transferase activity, did not induce it. The induction of mef(E) did not require macrolide efflux, but the affinity of macrolides for the ribosome determined the availability for efflux and pneumococcal susceptibility. The induction of mef(E)-mel expression by inducing macrolides appears to be based on specific interactions of the macrolide C-5 saccharide with the ribosome that alleviate transcriptional attenuation of mef(E)-mel.
Antimicrobial Agents and Chemotherapy 07/2011; 55(7):3413-22. · 4.84 Impact Factor
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ABSTRACT: Streptococcus pneumoniae type 2 pili are recently identified fimbrial structures extending from the bacterial surface and formed by polymers of the structural protein PitB. Intramolecular isopeptide bonds are a characteristic of the related pilus backbone protein Spy0128 of group A streptococci. Based on the identification of conserved residues in PitB, we predicted two intramolecular isopeptide bonds in PitB. Using a combination of tandem mass spectrometry and Edman sequencing, we show that these bonds were formed between Lys(63)-Asn(214) and Lys(243)-Asn(372) in PitB. Mutant proteins lacking the intramolecular isopeptide bonds retained the proteolytic stability observed with the wild type protein. However, absence of these bonds substantially decreased the melting temperature of the PitB-derivatives, indicating a stabilizing function of these bonds in PitB of the pneumococcal type 2 pilus.
Biochemical and Biophysical Research Communications 06/2011; 409(3):526-31. · 2.48 Impact Factor
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ABSTRACT: Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguinis are members of the Mitis group of streptococci and agents of oral biofilm, dental plaque and infective endocarditis, disease processes that involve bacteria-bacteria and bacteria-host interactions. Their close relative, the human pathogen S. pneumoniae uses pilus-islet 2 (PI-2)-encoded pili to facilitate adhesion to eukaryotic cells.
PI-2 pilus-encoding genetic islets were identified in S. oralis, S. mitis, and S. sanguinis, but were absent from other isolates of these species. The PI-2 islets resembled the genetic organization of the PI-2 islet of S. pneumoniae, but differed in the genes encoding the structural pilus proteins PitA and PitB. Two and three variants of pitA (a pseudogene in S. pneumoniae) and pitB, respectively, were identified that showed ≈20% difference in nucleotide as well as corresponding protein sequence. Species-independent combinations of pitA and pitB variants indicated prior intra- and interspecies horizontal gene transfer events. Polyclonal antisera developed against PitA and PitB of S. oralis type strain ATCC35037 revealed that PI-2 pili in oral streptococci were composed of PitA and PitB. Electronmicrographs showed pilus structures radiating >700 nm from the bacterial surface in the wild type strain, but not in an isogenic PI-2 deletion mutant. Anti-PitB-antiserum only reacted with pili containing the same PitB variant, whereas anti-PitA antiserum was cross-reactive with the other PitA variant. Electronic multilocus sequence analysis revealed that all PI-2-encoding oral streptococci were closely-related and cluster with non-PI-2-encoding S. oralis strains.
This is the first identification of PI-2 pili in Mitis group oral streptococci. The findings provide a striking example of intra- and interspecies horizontal gene transfer. The PI-2 pilus diversity provides a possible key to link strain-specific bacterial interactions and/or tissue tropisms with pathogenic traits in the Mitis group streptococci.
PLoS ONE 01/2011; 6(9):e25124. · 4.09 Impact Factor
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ABSTRACT: To define the prevalence of pilus islet 2 (PI-2)-encoded pili in Streptococcus pneumoniae in a geographically defined area, we examined 590 S. pneumoniae isolates from population-based surveillance of invasive pneumococcal disease in Atlanta, Georgia, USA, 1994-2006. In 2006, PI-2 was present in 21% of all invasive isolates, including serotypes 1 (100%), 7F (89%), 11A (21%), 19A (40%), and 19F (75%). Only serotype 19F is included in the 7-valent pneumococcal conjugate vaccine that is in use worldwide. In 1999, PI-2-containing isolates were of the same serotypes but accounted for only 3.6% of all invasive isolates. The increase of PI-2 in 2006 resulted predominantly from the emergence of serotype 19A isolates of sequence type 320 and the expansion of serotype 7F isolates. The increase in PI-2-containing isolates and the finding that isolates of all identified serotypes expressed highly conserved PI-2 pili supports their potential as a vaccine candidate.
Emerging Infectious Diseases 06/2010; 16(6):955-62. · 6.79 Impact Factor
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ABSTRACT: Macrolide resistance is a major concern in the treatment of Streptococcus pneumoniae. Inducible macrolide resistance in this pneumococcus is mediated by the efflux pump MefE/Mel. We show here that the human antimicrobial peptide LL-37 induces the mefE promoter and confers resistance to erythromycin and LL-37. Such induction may impact the efficacy of host defenses and of macrolide-based treatment of pneumococcal disease.
Antimicrobial Agents and Chemotherapy 05/2010; 54(8):3516-9. · 4.84 Impact Factor
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ABSTRACT: The important human pathogen Streptococcus pyogenes (group A streptococcus, GAS) initiates infection by pilus-mediated attachment to host tissue. Thus, the pilus is an excellent target for design of anti-infective strategies. The T3 pilus of GAS is composed of multiple covalently linked subunits of the T3 protein to which the two minor pilins, Cpa and OrfB, are covalently attached. Because the proteins of GAS pili do not contain either of the motifs required for pilus polymerization in other Gram-positive bacteria, we investigated the residues involved in their linkage. We show that linkage of Cpa to T3 by the sortase family transpeptidase SrtC2 requires the VPPTG motif in the cell wall-sorting signal of Cpa. We also demonstrate that K173 of T3 is required both for T3 polymerization and for attachment of Cpa to T3. Therefore, attachment of Cpa to K173 of a T3 subunit would block further addition of T3 subunits to this end of the growing pilus. This implies that Cpa is located exclusively at the pilus tip, a location supported by immunogold electron microscopy, and suggests that, as for well-studied pili on Gram-negative bacteria, the role of the pilus is to present the adhesin external to the bacterial capsule.
Molecular Microbiology 06/2009; 72(6):1379-94. · 5.01 Impact Factor
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ABSTRACT: Pili are a major surface feature of the human pathogen Streptococcus pyogenes (group A streptococcus [GAS]). The T3 pilus is composed of a covalently linked polymer of protein T3 (formerly Orf100 or Fct3) with an ancillary protein, Cpa, attached. A putative signal peptidase, SipA (also called LepA), has been identified in several pilus gene clusters of GAS. We demonstrate that the SipA2 allele of a GAS serotype M3 strain is required for synthesis of T3 pili. Heterologous expression in Escherichia coli showed that SipA2, along with the pilus backbone protein T3 and the sortase SrtC2, is required for polymerization of the T3 protein. In addition, we found that SipA2 is also required for linkage of the ancillary pilin protein Cpa to polymerized T3. Despite partial conservation of motifs of the type I signal peptidase family proteins, SipA lacks the highly conserved and catalytically important serine and lysine residues of these enzymes. Substitution of alanine for either of the two serine residues closest to the expected location of an active site serine demonstrated that these serine residues are both dispensable for T3 polymerization. Therefore, it seems unlikely that SipA functions as a signal peptidase. However, a T3 protein mutated at the P-1 position of the signal peptide cleavage site (alanine to arginine) was unstable in the presence of SipA2, suggesting that there is an interaction between SipA and T3. A possible chaperone-like function of SipA2 in T3 pilus formation is discussed.
Journal of bacteriology 02/2008; 190(2):527-35. · 3.94 Impact Factor
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ABSTRACT: Bacteria attach to their appropriate environmental niche by using adhesins. To maximize their contact with the environment, adhesins are often present on the ends of long hairlike structures called pili. Recently, attention has focused on pili of Gram-positive bacteria because they may be vaccine candidates in important human pathogens. These pili differ from the well-studied pili of Gram-negative bacteria because their subunits are covalently linked, they do not require specific chaperones for assembly, and the tip protein (likely to be the adhesin) is not required to initiate formation of the pilus structure. In Gram-positive bacteria, the genes for pili occur in clusters, which may constitute mobile genetic elements. These clusters include the transpeptidase(s) of the sortase family that is/are required for polymerization of the subunit proteins. However, efficient covalent attachment of the completed pilus structure to the cell wall is accomplished, in cases where this has been studied, by the 'housekeeping' sortase, which is responsible for attachment to the peptidoglycan of most surface proteins containing cell wall sorting signals. This enzyme is encoded elsewhere on the genome. Because pili of Gram-positive bacteria have not been extensively investigated yet, we hope that this MicroReview will help to pinpoint the areas most in need of further study.
Molecular Microbiology 11/2006; 62(2):320-30. · 5.01 Impact Factor
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ABSTRACT: 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.
Journal of Bacteriology 04/2006; 188(5):1959-68. · 3.83 Impact Factor
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ABSTRACT: The ciaR-ciaH system is one of 13 two-component signal-transducing systems of the human pathogen Streptococcus pneumoniae. Mutations in the histidine protein kinase CiaH confer increased resistance to beta-lactam antibiotics and interfere with the development of genetic competence. In order to identify the genes controlled by the cia system, the cia regulon, DNA fragments targeted by the response regulator CiaR were isolated from restricted chromosomal DNA using the solid-phase DNA binding assay and analyzed by hybridization to an oligonucleotide microarray representing the S. pneumoniae genome. A set of 18 chromosomal regions containing 26 CiaR target sites were detected and proposed to represent the minimal cia regulon. The putative CiaR target loci included genes important for the synthesis and modification of cell wall polymers, peptide pheromone and bacteriocin production, and the htrA-spo0J region. In addition, the transcription profile of cia loss-of-function mutants and those with an apparent activated cia system representing the off and on states of the regulatory system were analyzed. The transcript analysis confirmed the cia-dependent expression of seven putative target loci and revealed three additional cia-regulated loci. Five putative target regions were silent under all conditions, and for the remaining three regions, no cia-dependent expression could be detected. Furthermore, the competence regulon, including the comCDE operon required for induction of competence, was completely repressed by the cia system.
Journal of Bacteriology 02/2003; 185(1):60-70. · 3.83 Impact Factor
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ABSTRACT: New mechanisms for beta-lactam resistance independent on the target penicillin-binding proteins were detected in beta-lactam-resistant laboratory mutants of Streptococcus pneumoniae. The link between mutations in the histidine protein kinase CiaH and phenotypic expression of cefotaxime resistance suggests that the cell is able to monitor the integrity of the cell wall and in emergency cases such as during the action of beta-lactams can counteract such danger. At least one ciaH mutation Thr230 > Pro is likely to affect its phosphatase activity resulting in elevated phosphorylation of CiaR, the cognate response regulator, but other CiaH-independent signaling pathways may also result in CiaR phosphorylation. Mutants in CiaH, either alone or in combination with a mutated penicillin-binding protein 2x(PBP2x) fail to develop genetic competence. In all cases complementation of this phenotype was observed upon addition of the competence inducing pheromone peptide CSP, the processed product of the comC gene. This indicates that the cia system is part of a regulatory network that includes another two component system comDE. The DNA binding property of CiaR and ComE were exploited to isolate specifically interacting DNA fragments as a first step to identify genes targeted by individual response regulators.
Journal of Molecular Microbiology and Biotechnology 05/2002; 4(3):211-6. · 1.95 Impact Factor
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ABSTRACT: The β-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 β-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 β-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 β-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 β-lactam treatment counteract the effects caused by the inhibition of penicillin-binding proteins.
Molecular Microbiology 07/1999; 33(4):673 - 678. · 5.01 Impact Factor
