RESUMO
The opportunistic pathogen Streptococcus mitis possesses, like other members of the Mitis group of viridans streptococci, phosphorylcholine (P-Cho)-containing teichoic acids (TAs) in its cell wall. Bioinformatic analyses predicted the presence of TAs that are almost identical with those identified in the pathogen Streptococcus pneumoniae, but a detailed analysis of S. mitis lipoteichoic acid (LTA) was not performed to date. Here, we determined the structures of LTA from two S. mitis strains, the high-level beta-lactam and multiple antibiotic resistant strain B6 and the penicillin-sensitive strain NCTC10712. In agreement with bioinformatic predictions, we found that the structure of one LTA (type IV) was like pneumococcal LTA, except the exchange of a glucose moiety with a galactose within the repeating units. Further genome comparisons suggested that the majority of S. mitis strains should contain the same type IV LTA as S. pneumoniae, providing a more complete understanding of the biosynthesis of these P-Cho-containing TAs in members of the Mitis group of streptococci. Remarkably, we observed besides type IV LTA, an additional polymer belonging to LTA type I in both investigated S. mitis strains. This LTA consists of ß-galactofuranosyl-(1,3)-diacylglycerol as glycolipid anchor and a poly-glycerol-phosphate chain at the O-6 position of the furanosidic galactose. Hence, these bacteria are capable of synthesizing two different LTA polymers, most likely produced by distinct biosynthesis pathways. Our bioinformatics analysis revealed the prevalence of the LTA synthase LtaS, most probably responsible for the second LTA version (type I), among S. mitis and Streptococcus pseudopneumoniae strains.
Assuntos
Streptococcus mitis , Ácidos Teicoicos , Lipopolissacarídeos/química , Streptococcus mitis/genética , Streptococcus mitis/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Ácidos Teicoicos/químicaRESUMO
Streptococcus pneumoniae is an ovoid-shaped Gram-positive bacterium that grows by carrying out peripheral and septal peptidoglycan (PG) synthesis, analogous to model bacilli, such as Escherichia coli and Bacillus subtilis In the model bacilli, FtsZ and FtsA proteins assemble into a ring at midcell and are dedicated to septal PG synthesis but not peripheral PG synthesis; hence, inactivation of FtsZ or FtsA results in long filamentous cells unable to divide. Here, we demonstrate that FtsA and FtsZ colocalize at midcell in S. pneumoniae and that partial depletion of FtsA perturbs septum synthesis, resulting in elongated cells with multiple FtsZ rings that fail to complete septation. Unexpectedly, complete depletion of FtsA resulted in the delocalization of FtsZ rings and ultimately cell ballooning and lysis. In contrast, depletion or deletion of gpsB and sepF, which in B. subtilis are synthetically lethal with ftsA, resulted in enlarged and elongated cells with multiple FtsZ rings, with deletion of sepF mimicking partial depletion of FtsA. Notably, cell ballooning was not observed, consistent with later recruitment of these proteins to midcell after Z-ring assembly. The overproduction of FtsA stimulates septation and suppresses the cell division defects caused by the deletion of sepF and gpsB under some conditions, supporting the notion that FtsA shares overlapping functions with GpsB and SepF at later steps in the division process. Our results indicate that, in S. pneumoniae, both GpsB and SepF are involved in septal PG synthesis, whereas FtsA and FtsZ coordinate both peripheral and septal PG synthesis and are codependent for localization at midcell.IMPORTANCEStreptococcus pneumoniae (pneumococcus) is a clinically important human pathogen for which more therapies against unexploited essential targets, like cell growth and division proteins, are needed. Pneumococcus is an ovoid-shaped Gram-positive bacterium with cell growth and division properties that have important distinctions from those of rod-shaped bacteria. Gaining insights into these processes can thus provide valuable information to develop novel antimicrobials. Whereas rods use distinctly localized protein machines at different cellular locations to synthesize peripheral and septal peptidoglycans, we present evidence that S. pneumoniae organizes these two machines at a single location in the middle of dividing cells. Here, we focus on the properties of the actin-like protein FtsA as an essential orchestrator of peripheral and septal growth in this bacterium.
RESUMO
Penicillin-resistant Streptococcus pneumoniae strains are found at high rates in Romania and Iran. The mosaic structure of PBP2x was investigated in 9 strains from Iran and in 15 strains from Romania to understand their evolutionary history. Mutations potentially important for ß-lactam resistance were identified by comparison of the PBP2x sequences with the sequence of the related PBP2x of reference penicillin-sensitive S. mitis strains. Two main PBP2x mosaic gene families were recognized. Eight Iranian strains expressed PBP2x variants in group 1, which had a mosaic block highly related to PBP2x of the Spain23F-1 clone, which is widespread among international penicillin-resistant S. pneumoniae clones. A second unique PBP2x group was observed in Romanian strains; furthermore, three PBP2x single mosaic variants were found. Sequence blocks of penicillin-sensitive strain S. mitis 658 were common among PBP2x variants from strains from both countries. Each PBP2x group contained specific signature mutations within the transpeptidase domain, documenting the existence of distinct mutational pathways for the development of penicillin resistance.
Assuntos
Antibacterianos/farmacologia , Mosaicismo , Resistência às Penicilinas/genética , Proteínas de Ligação às Penicilinas/genética , Penicilinas/farmacologia , Streptococcus pneumoniae/genética , Idoso , Sequência de Aminoácidos , Criança , Pré-Escolar , Células Clonais , Feminino , Expressão Gênica , Humanos , Lactente , Irã (Geográfico) , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Modelos Moleculares , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/metabolismo , Polimorfismo Genético , Romênia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , Streptococcus mitis/efeitos dos fármacos , Streptococcus mitis/genética , Streptococcus mitis/isolamento & purificação , Streptococcus mitis/metabolismo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pneumoniae/metabolismo , Adulto JovemRESUMO
The Streptococcus pneumoniae clone Hungary19A-6 expresses unusually high levels of ß-lactam resistance, which is in part due to mutations in the MurM gene, encoding a transferase involved in the synthesis of branched peptidoglycan. Moreover, it contains the allele ciaH232, encoding the histidine kinase CiaH (M. Müller, P. Marx, R. Hakenbeck, and R. Brückner, Microbiology 157:3104-3112, 2011, https://doi.org/10.1099/mic.0.053157-0). High-level penicillin resistance primarily requires the presence of low-affinity (mosaic) penicillin binding protein (PBP) genes, as, for example, in strain Hu17, a closely related member of the Hungary19A-6 lineage. Interestingly, strain Hu15 is ß-lactam sensitive due to the absence of mosaic PBPs. This unique situation prompted us to investigate the development of cefotaxime resistance in transformation experiments with genes known to play a role in this phenotype, pbp2x, pbp1a, murM, and ciaH, and penicillin-sensitive recipient strains R6 and Hu15. Characterization of phenotypes, peptidoglycan composition, and CiaR-mediated gene expression revealed several novel aspects of penicillin resistance. The murM gene of strain Hu17 (murMHu17), which is highly similar to murM of Streptococcus mitis, induced morphological changes which were partly reversed by ciaH232. murMHu17 conferred cefotaxime resistance only in the presence of the pbp2x of strain Hu17 (pbp2xHu17). The ciaH232 allele contributed to a remarkable increase in cefotaxime resistance in combination with pbp2xHu17 and pbp1a of strain Hu17 (pbp1aHu17), accompanied by higher levels of expression of CiaR-regulated genes, documenting that ciaH232 responds to PBP1aHu17-mediated changes in cell wall synthesis. Most importantly, the proportion of branched peptides relative to the proportion of linear muropeptides increased in cells containing mosaic PBPs, suggesting an altered enzymatic activity of these proteins.
Assuntos
Proteínas de Ligação às Penicilinas/metabolismo , Penicilinas/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Hungria , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Peptídeo Sintases/metabolismo , Peptidoglicano/metabolismo , Fenótipo , Sorogrupo , Streptococcus pneumoniae/enzimologia , beta-Lactamas/metabolismoRESUMO
The transpeptidase activity of the essential penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae is believed to be important for murein biosynthesis required for cell division. To study the molecular mechanism driving localization of PBP2x in live cells, we constructed a set of N-terminal GFP-PBP2x fusions under the control of a zinc-inducible promoter. The ectopic fusion protein localized at mid-cell. Cells showed no growth defects even in the absence of the genomic pbp2x, demonstrating that GFP-PBP2x is functional. Depletion of GFP-PBP2x resulted in severe morphological alterations, confirming the essentiality of PBP2x and demonstrating that PBP2x is required for cell division and not for cell elongation. A genetically or antibiotic inactivated GFP-PBP2x still localized at septal sites. Remarkably, the same was true for a GFP-PBP2x derivative containing a deletion of the central transpeptidase domain, although only in the absence of the protease/chaperone HtrA. Thus localization is independent of the catalytic transpeptidase domain but requires the C-terminal PASTA domains, identifying HtrA as targeting GFP-PBP2x derivatives. Finally, PBP2x was positioned at the septum similar to PBP1a and the PASTA domain containing StkP protein, confirming that PBP2x is a key element of the divisome complex.
Assuntos
Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Sinais Direcionadores de Proteínas/genética , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/enzimologia , Divisão Celular , Forma Celular , Chlamydophila pneumoniae , Análise Mutacional de DNA , Genes Essenciais , Manutenção , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/fisiologiaRESUMO
The human pathogen Streptococcus pneumoniae has been treated for decades with ß-lactam antibiotics. Its resistance is now widespread, mediated by the expression of mosaic variants of the target enzymes, the penicillin-binding proteins (PBPs). Understanding the mode of action of ß-lactams, not only in molecular detail but also in their physiological consequences, will be crucial to improving these drugs and any counterresistances. In this work, we investigate the piperacillin paradox, by which this ß-lactam selects primarily variants of PBP2b, whereas its most reactive target is PBP2x. These PBPs are both essential monofunctional transpeptidases involved in peptidoglycan assembly. PBP2x participates in septal synthesis, while PBP2b functions in peripheral elongation. The formation of the "lemon"-shaped cells induced by piperacillin treatment is consistent with the inhibition of PBP2x. Following the examination of treated and untreated cells by electron microscopy, the localization of the PBPs by epifluorescence microscopy, and the determination of the inhibition time course of the different PBPs, we propose a model of peptidoglycan assembly that accounts for the piperacillin paradox.
Assuntos
Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Piperacilina/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Resistência beta-Lactâmica , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Testes de Sensibilidade Microbiana , Terapia de Alvo Molecular , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/fisiologia , Resistência beta-Lactâmica/efeitos dos fármacosRESUMO
Heteroresistance to penicillin in Streptococcus pneumoniae is the ability of subpopulations to grow at a higher antibiotic concentration than expected from the MIC. This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary ß-lactam resistance determinants, penicillin-binding protein 2b (PBP2b) and PBP2x, and the secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from the heteroresistant strain Spain(23F) 2349 in the nonheteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found that pbp2x, but not pbp2b or pbp1a alone, conferred heteroresistance to R6. However, a change of pbp2x expression was not observed, and therefore, expression does not correlate with an increased proportion of resistant subpopulations. In addition, the influence of the CiaRH system, mediating PBP-independent ß-lactam resistance, was assessed by PAP on ciaR disruption mutants but revealed no heteroresistant phenotype. We also showed that the highly resistant subpopulations (HOM*) of transformants containing low-affinity pbp2x undergo an increase in resistance upon selection on penicillin plates that partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins encoded by pstS, phoU, pstB, and pstC in these highly resistant subpopulations. In conclusion, the presence of low-affinity pbp2x enables certain pneumococcal colonies to survive in the presence of ß-lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaptation to antibiotic stress.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Resistência às Penicilinas/genética , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Infecções Pneumocócicas/microbiologia , Proteômica/métodos , Reação em Cadeia da Polimerase em Tempo Real , Streptococcus pneumoniae/crescimento & desenvolvimento , Transformação BacterianaRESUMO
Streptococcus oralis, a commensal species of the human oral cavity, belongs to the Mitis group of streptococci, which includes one of the major human pathogens as well, S. pneumoniae. We report here the first complete genome sequence of this species. S. oralis Uo5, a high-level penicillin- and multiple-antibiotic-resistant isolate from Hungary, is competent for genetic transformation under laboratory conditions. Comparative and functional genomics of Uo5 will be important in understanding the evolution of pathogenesis among Mitis streptococci and their potential to engage in interspecies gene transfer.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Análise de Sequência de DNA , Streptococcus oralis/genética , Farmacorresistência Bacteriana Múltipla , Humanos , Hungria , Dados de Sequência Molecular , Boca , Streptococcus oralis/efeitos dos fármacos , Streptococcus oralis/isolamento & purificação , Transformação BacterianaRESUMO
Reduced amounts of the essential penicillin-binding protein 2x (PBP2x) were detected in two cefotaxime-resistant Streptococcus pneumoniae laboratory mutants C405 and C606. These mutants contain two or four mutations in the penicillin-binding domain of PBP2x, respectively. The transcription of the pbp2x gene was not affected in both mutants; thus, the reduced PBP2x amounts were likely due to post-transcriptional regulation. The mutants carry a mutation in the histidine protein kinase gene ciaH, resulting in enhanced gene expression mediated by the cognate response regulator CiaR. Deletion of htrA, encoding a serine protease regulated by CiaR, or inactivation of HtrA proteolytic activity showed that HtrA is indeed responsible for PBP2x degradation in both mutants, and that this affects ß-lactam resistance. Depletion of the PBP2xC405 in different genetic backgrounds confirmed that HtrA degrades PBP2xC405. A GFP-PBP2xC405 fusion protein still localized at the septum in the absence of HtrA. The complementation studies in HtrA deletion strains showed that HtrA can be overexpressed in pneumococcal cells to specific levels, depending on the genetic background. Quantitative Western blotting revealed that the PBP2x amount in C405 strain was less than 20% compared to parental strain, suggesting that PBP2x is an abundant protein in S. pneumoniae R6 strain.
RESUMO
Penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae represents a primary resistance determinant for beta-lactams, and low-affinity PBP2x variants can easily be selected with cefotaxime. Penicillin-resistant clinical isolates of S. pneumoniae frequently contain in their mosaic PBP2x the mutation T338A adjacent to the active site S337, and T338P as well as T338G substitutions are also known. Site-directed mutagenesis has now documented that a single point mutation at position T338 confers selectable levels of beta-lactam resistance preferentially to oxacillin. Despite the moderate impact on beta-lactam susceptibility, the function of the PBP2x mutants appears to be impaired, as can be documented in the absence of a functional CiaRH regulatory system, resulting in growth defects and morphological changes. The combination of low-affinity PBP2x and PBP1a encoded by mosaic genes is known to result in high cefotaxime resistance. In contrast, introduction of a mosaic pbp1a into the PBP2x(T338G) mutant did not lead to increased resistance. However, the mosaic PBP1a gene apparently complemented the PBP2x(T338G) defect, since Cia mutant derivatives grew normally. The data support the view that PBP2x and PBP1a interact with each other on some level and that alterations of both PBPs in resistant clinical isolates have evolved to ensure cooperation between both proteins.
Assuntos
Proteínas de Ligação às Penicilinas/genética , Mutação Puntual , Streptococcus pneumoniae/química , Streptococcus pneumoniae/genética , Treonina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Sítios de Ligação/genética , Genes Bacterianos , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Resistência às Penicilinas/genética , Penicilinas/metabolismo , Ligação Proteica , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pneumoniae/metabolismo , Transformação BacterianaRESUMO
The two recently characterized Streptococcus pneumoniae strains--R6Chi and R6Cho(-)--that have lost the unique auxotrophic requirement of this bacterial species for choline differ in their mechanisms of choline independence. In strain R6Chi the mechanism is caused by a point mutation in tacF, a gene that is part of the pneumococcal lic2 operon, which is essential for growth and survival of the bacteria. Cultures of lic2 mutants of the encapsulated strain D39Chi growing in choline-containing medium formed long chains, did not autolyze, had no choline in their cell wall, and were completely avirulent in the mouse intraperitoneal model. In contrast, while the Cho(-) strain carried a complete pneumococcal lic2 operon and had no mutations in the tacF gene, deletion of the entire lic2 operon had no effect on the growth or phenotype of strain Cho(-). These observations suggest that the biochemical functions normally dependent on determinants of the pneumococcal lic2 operon may also be carried out in strain Cho(-) by a second set of genetic elements imported from Streptococcus oralis, the choline-independent streptococcal strain that served as the DNA donor in the heterologous transformation event that produced strain R6Cho(-). The identification in R6Cho(-) of a large (20-kb) S. oralis DNA insert carrying both tacF and licD genes confirms this prediction and suggests that these heterologous elements may represent a "backup" system capable of catalyzing P-choline incorporation and export of teichoic acid chains under conditions in which the native lic2 operon is not functional.
Assuntos
Colina/metabolismo , Streptococcus pneumoniae/metabolismo , Animais , Parede Celular/metabolismo , Feminino , Inativação Gênica , Genes Bacterianos/genética , Camundongos , Modelos Genéticos , Óperon/genética , Especificidade da Espécie , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Transcrição Gênica , Virulência/genéticaRESUMO
The draft genome sequences of two multiple-antibiotic-resistant Streptococcus pneumoniae isolates from Hungary, Hu15 and Hu17, are reported here. Strain Hu15 is penicillin susceptible, whereas Hu17 is a high-level-penicillin-resistant strain. Both isolates belong to the serotype 19A sequence type 226, a single-locus variant (in the ddl locus) of the Hungary19A-6 clone.
RESUMO
Penicillin-binding proteins (PBPs) are membrane-associated enzymes, which are involved in the last two steps of peptidoglycan biosynthesis, and some of them are key players in cell division. Furthermore, they are targets of ß-lactams, the most widely used antibiotics. Nevertheless, very little is known about the expression and regulation of PBP genes. Using transcriptional mapping, we now determined the promoter regions of PBP genes from the laboratory strain Streptococcus pneumoniae R6 and examined the expression profile of these six promoters. The extended -10 region is highly conserved and complies with a σ(A)-type promoter consensus sequence. In contrast, the -35 region is poorly conserved, indicating the possibility for differential PBP regulation. All PBP promoters were constitutively expressed and highly active during the exponential and early stationary growth phase. However, the individual expression of PBP promoters varied approximately fourfold, with pbp1a being the highest and pbp3 the lowest. Furthermore, the deletion of one nucleotide in the spacer region of the PBP3 promoter reduced pbp3 expression â¼10-fold. The addition of cefotaxime above the minimal inhibitory concentration (MIC) did not affect PBP expression in the penicillin-sensitive R6 strain. No evidence for regulation of S. pneumoniae PBP genes was obtained.
Assuntos
Regulação Bacteriana da Expressão Gênica , Resistência às Penicilinas/genética , Proteínas de Ligação às Penicilinas/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Antibacterianos/farmacologia , Sequência de Bases , Cefotaxima/farmacologia , Sequência Conservada , Testes de Sensibilidade Microbiana , Mutação , Proteínas de Ligação às Penicilinas/metabolismo , Penicilinas/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Streptococcus pneumoniaeRESUMO
Viridans streptococci were obtained from primates (great apes, rhesus monkeys, and ring-tailed lemurs) held in captivity, as well as from free-living animals (chimpanzees and lemurs) for whom contact with humans is highly restricted. Isolates represented a variety of viridans streptococci, including unknown species. Streptococcus oralis was frequently isolated from samples from great apes. Genotypic methods revealed that most of the strains clustered on separate lineages outside the main cluster of human S. oralis strains. This suggests that S. oralis is part of the commensal flora in higher primates and evolved prior to humans. Many genes described as virulence factors in Streptococcus pneumoniae were present also in other viridans streptococcal genomes. Unlike in S. pneumoniae, clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) gene clusters were common among viridans streptococci, and many S. oralis strains were type PI-2 (pilus islet 2) variants. S. oralis displayed a remarkable diversity of genes involved in the biosynthesis of peptidoglycan (penicillin-binding proteins and MurMN) and choline-containing teichoic acid. The small noncoding cia-dependent small RNAs (csRNAs) controlled by the response regulator CiaR might contribute to the genomic diversity, since we observed novel genomic islands between duplicated csRNAs, variably present in some isolates. All S. oralis genomes contained a ß-N-acetyl-hexosaminidase gene absent in S. pneumoniae, which in contrast frequently harbors the neuraminidases NanB/C, which are absent in S. oralis. The identification of S. oralis-specific genes will help us to understand their adaptation to diverse habitats. IMPORTANCE Streptococcus pneumoniae is a rare example of a human-pathogenic bacterium among viridans streptococci, which consist of commensal symbionts, such as the close relatives Streptococcus mitis and S. oralis. We have shown that S. oralis can frequently be isolated from primates and a variety of other viridans streptococci as well. Genes and genomic islands which are known pneumococcal virulence factors are present in S. oralis and S. mitis, documenting the widespread occurrence of these compounds, which encode surface and secreted proteins. The frequent occurrence of CRISP-Cas gene clusters and a surprising variation of a set of small noncoding RNAs are factors to be considered in future research to further our understanding of mechanisms involved in the genomic diversity driven by horizontal gene transfer among viridans streptococci.
RESUMO
Members of the Mitis group of streptococci possess teichoic acids (TAs) as integral components of their cell wall that are unique among Gram-positive bacteria. Both, lipoteichoic (LTA) and wall teichoic acid, are formed by the same biosynthetic pathway, are of high complexity and contain phosphorylcholine (P-Cho) residues. These residues serve as anchors for choline-binding proteins (CBPs), some of which have been identified as virulence factors of the human pathogen Streptococcus pneumoniae. We investigated the LTA structure of its close relative Streptococcus oralis. Our analysis revealed that S. oralis Uo5 LTA has an overall architecture similar to pneumococcal LTA (pnLTA) and can be considered as a subtype of type IV LTA. Its structural complexity is even higher than that of pnLTA and its composition differs in number and type of carbohydrate moieties, inter-residue connectivities and especially the P-Cho substitution pattern. Here, we report the occurrence of a saccharide moiety substituted with two P-Cho residues, which is unique as yet in bacterial derived surface carbohydrates. Finally, we could link the observed important structural variations between S. oralis and S. pneumoniae LTA to the divergent enzymatic repertoire for their TA biosynthesis.
Assuntos
Lipopolissacarídeos/química , Fosforilcolina/química , Streptococcus oralis/química , Ácidos Teicoicos/química , Vias Biossintéticas , Parede Celular/química , Parede Celular/metabolismo , Hidrazinas/farmacologia , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/metabolismo , Espectrometria de Massas , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilcolina/metabolismo , Streptococcus oralis/metabolismo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/metabolismo , Ácidos Teicoicos/biossíntese , Ácidos Teicoicos/metabolismoRESUMO
Streptococcus pneumoniae penicillin-binding protein 2x (PBP2x) is an enzyme involved in the last stages of peptidoglycan assembly and essential for bacterial growth and survival. PBP2x localizes to the division site, a process that depends on its Penicillin-Binding Protein And Serine-Threonine-kinase Associated (PASTA) domains, which was previously demonstrated via GFP-PBP2x in living cells. During this study a mutant strain was isolated in which the GFP-PBP2x fusion protein did not localize at division sites and it contained reduced amounts of the full-length GFP-PBP2x. We now show that this defect is due to a point mutation within the C-terminal PASTA2 domain of PBP2x. The mutant protein was analyzed in detail in terms of beta-lactam binding, functionality, and localization in live cells. We demonstrate that the mutation affects the GFP-tagged PBP2x variant severely and renders it susceptible to the protease/chaperone HtrA.
Assuntos
Proteínas de Bactérias/genética , Parede Celular/genética , Proteínas de Ligação às Penicilinas/genética , Proteínas Recombinantes de Fusão/genética , Streptococcus pneumoniae/genética , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/enzimologia , Expressão Gênica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/biossíntese , Proteínas Periplásmicas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/metabolismo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , beta-Lactamas/farmacologiaRESUMO
Alterations in the target enzymes for ß-lactam antibiotics, the penicillin-binding proteins (PBPs), have been recognized as a major resistance mechanism in Streptococcus pneumoniae. Mutations in PBPs that confer a reduced affinity to ß-lactams have been identified in laboratory mutants and clinical isolates, and document an astounding variability of sites involved in this phenotype. Whereas point mutations are selected in the laboratory, clinical isolates display a mosaic structure of the affected PBP genes, the result of interspecies gene transfer and recombination events. Depending on the selective ß-lactam, different combinations of PBP genes and mutations within are involved in conferring resistance, and astoundingly in non-PBP genes as well.
Assuntos
Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/metabolismo , Resistência beta-Lactâmica , beta-Lactamas/farmacologia , Animais , Humanos , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Streptococcus pneumoniae/genéticaRESUMO
The cell wall of Streptococcus pneumoniae contains an unusually complex wall teichoic acid (WTA), which has identical repeating units as the membrane-anchored lipoteichoic acid (LTA). Both polymers share a common cytoplasmic pathway of precursor synthesis, but several TA enzymes have remained elusive. Bioinformatic analysis of the genome of various pneumococcal strains, including choline-independent mutant strains, has allowed us to identify the missing TA genes. We present here the deduced complete pathways of WTA and LTA synthesis in S. pneumoniae and point to the variations occurring in different pneumococcal strains and in closely related species such as Streptococcus oralis and Streptococcus mitis.
Assuntos
Genoma Bacteriano , Streptococcus mitis/genética , Streptococcus oralis/genética , Streptococcus pneumoniae/genética , Ácidos Teicoicos/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Parede Celular/química , Parede Celular/metabolismo , Colina/metabolismo , Colina/farmacologia , Biologia Computacional , Genômica , Dados de Sequência Molecular , Streptococcus mitis/metabolismo , Streptococcus oralis/metabolismo , Streptococcus pneumoniae/metabolismoRESUMO
The presence of new Streptococcus pneumoniae clones in dead wild chimpanzees from the Taï National Park, Côte d'Ivoire, with previous respiratory problems has been demonstrated recently by DNA sequence analysis from samples obtained from the deceased apes. In order to broadenour understanding on the relatedness of these pneumococcal clones to those from humans, the gene locus responsible for biosynthesis of the capsule polysaccharide (CPS) has now been characterized. DNA sequence analysis of PCR fragments identified a cluster named cps3(Taï) containing the four genes typical for serotype 3 CPS, but lacking a 5'-region of ≥2 kb which is degenerated in other cps3 loci and not required for type 3 biosynthesis. CPS3 is composed of a simple disaccharide repeat unit comprising glucose and glucuronic acid (GlcUA). The two genes ugd responsible for GlcUA synthesis and wchE encoding the type 3 synthase are essential for CPS3 biosynthesis, whereas both, galU and the 3'-truncated gene pgm are not required due to the presence of homologues elsewhere in the genome. The DNA sequence of cps3(Taï) diverged considerably from those of other cps3 loci. Also, the gene pgm(Taï) represents a full length version with a nonsense mutation at codon 179. The two genes ugd(Taï) and wchE(Taï) including the promoter region were transformed into a nonencapsulated laboratory strain S. pneumoniae R6. Transformants which expressed type 3 capsule polysaccharide were readily obtained, documenting that the gene products are functional. In summary, the data indicate that cps3(Taï) evolved independent from other cps3 loci, suggesting the presence of specialized serotype 3 S. pneumoniae clones endemic to the Taï National Park area.