Pneumococcal biofilms.

Over 60% of bacterial infections (and up to 80% of chronic infections) are currently considered to involve microbial growth in biofilms. This peculiar form of life poses an array of problems in human clinical practice, from infections associated with the implant of prosthetic devices and dental plaque formation to diseases such as cystic fibrosis, otitis media, and endocarditis. Biofilms are also at the basis of a variety of problems in industry. This report describes the biofilms produced by Streptococcus pneumoniae. This bacterium often colonizes the upper airways in humans as a normal commensal, yet it may spread to other areas of the body, causing otitis media, pneumonia, or invasive diseases such as bacteremia and meningitis. The capacity of S. pneumoniae to form biofilms had not been explored until recently. Several newly developed in vitro systems have allowed to test the capacity of S. pneumoniae to form biofilms, and to analyze the influence of several factors, including DNA and proteins-which play a role in the virulence of this "supergerm" in the formation and development of biofilms. In this brief review, we update the knowledge available on pneumococcal biofilm formation and the unusual features of this structure.

Pneumococcal biofilms

Over 60% of bacterial infections (and up to 80% of chronic infections) are currently considered to involve microbial growth in biofilms. This peculiar form of life poses an array of problems in human clinical practice, from infections associated with the implant of prosthetic devices and dental plaque formation to diseases such as cystic fibrosis, otitis media, and endocarditis. Biofilms are also at the basis of a variety of problems in industry. This report describes the biofilms produced by Streptococcus pneumoniae. This bacterium often colonizes the upper airways in humans as a normal commensal, yet it may spread to other areas of the body, causing otitis media, pneumonia, or invasive diseases such as bacteremia and meningitis. The capacity of S. pneumoniae to form biofilms had not been explored until recently. Several newly developed in vitro systems have allowed to test the capacity of S. pneumoniae to form biofilms, and to analyze the influence of several factors, including DNA and proteins-which play a role in the virulence of this «supergerm» in the formation and development of biofilms. In this brief review, we update the knowledge available on pneumococcal biofilm formation and the unusual features of this structure (AU)

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In vitro interactions of LytA, the major pneumococcal autolysin, with two bacteriophage lytic enzymes (Cpl-1 and Pal), cefotaxime and moxifloxacin against antibiotic-susceptible and -resistant Streptococcus pneumoniae strains.

OBJECTIVES: In an innovative therapeutic exploitation against antibiotic-resistant Streptococcus pneumoniae, here we have evaluated the in vitro activity of a purified bacterially-encoded cell wall lytic enzyme, LytA (the major pneumococcal autolysin), and compared it with those of Cpl-1 and Pal (pneumococcal phage lytic enzymes) and two antibiotics versus four pneumococcal strains. METHODS: Two serotype 3, penicillin-susceptible strains and two penicillin-resistant (serotypes 19F and 19A, respectively) S. pneumoniae clinical isolates were used. The effect of several combinations of lytic enzymes and antibiotics (cefotaxime and moxifloxacin) was studied by chequerboard and time-kill assays, the latter at concentrations of 0.25 x MIC. RESULTS: LytA was more active than Cpl-1 and Pal. By the chequerboard technique, the combination of LytA and cefotaxime was synergistic for one of the two cefotaxime-resistant strains studied. The combined use of Cpl-1 and Pal was synergistic for three of the four strains, as was Cpl-1 with antibiotics for two of the three strains studied. In the time-kill assays, after 5 h of exposure to LytA, Cpl-1 or Pal, the mean differences in colony counts versus controls were -3.55, -2.66 and -2.71 log(10) cfu/mL, respectively. The combination of LytA/Pal reduced the bacterial inoculum >2 log units for three of the four strains. LytA combined with cefotaxime or moxifloxacin achieved >3 log units decrease for the strains tested. Particularly, a strong synergism was observed with LytA/cefotaxime for one cefotaxime-resistant meningeal strain. LytA/moxifloxacin was synergistic for the quinolone-resistant strain when tested by time-kill methodology, and just close to synergistic (fractional inhibitory concentration index of 0.58) by the chequerboard technique. Antagonism was not observed for any combination when assayed by either method. CONCLUSIONS: LytA, Cpl-1 or Pal, alone or in combination, might prove to be effective in combination therapy, as well as in monotherapy against S. pneumoniae. These results suggest avenues of research to study the cell wall lytic enzymes as anti-pneumococcal therapeutic agents.

Pneumococcal LytA autolysin, a potent therapeutic agent in experimental peritonitis-sepsis caused by highly beta-lactam-resistant Streptococcus pneumoniae.

The in vitro and in vivo antipneumococcal activities of the main pneumococcal autolysin (LytA) and Cpl-1, a lysozyme encoded by phage Cp-1, were studied. Intraperitoneal therapy with LytA or high-dose Cpl-1 remarkably reduced peritoneal bacterial counts (>5 log(10) CFU/ml) compared with those for the controls. After intravenous injection, LytA was the most effective treatment.

Key role of amino acid residues in the dimerization and catalytic activation of the autolysin LytA, an important virulence factor in Streptococcus pneumoniae.

LytA, the main autolysin of Streptococcus pneumoniae, was the first member of the bacterial N-acetylmuramoyl-l-alanine amidase (NAM-amidase) family of proteins to be well characterized. This autolysin degrades the peptidoglycan bonds of pneumococcal cell walls after anchoring to the choline residues of the cell wall teichoic acids via its choline-binding module (ChBM). The latter is composed of seven repeats (ChBRs) of approximately 20 amino acid residues. The translation product of the lytA gene is the low-activity E-form of LytA (a monomer), which can be "converted" (activated) in vitro by choline into the fully active C-form at low temperature. The C-form is a homodimer with a boomerang-like shape. To study the structural requirements for the monomer-to-dimer modification and to clarify whether "conversion" is synonymous with dimerization, the biochemical consequences of replacing four key amino acid residues of ChBR6 and ChBR7 (the repeats involved in dimer formation) were determined. The results obtained with a collection of 21 mutated NAM-amidases indicate that Ile-315 is a key amino acid residue in both LytA activity and folding. Amino acids with a marginal position in the solenoid structure of the ChBM were of minor influence in dimer stability; neither the size, polarity, nor aromatic nature of the replacement amino acids affected LytA activity. In contrast, truncated proteins were drastically impaired in their activity and conversion capacity. The results indicate that dimerization and conversion are different processes, but they do not answer the questions of whether conversion can only be achieved after a dimer formation step.

Isolation and characterization of a new plasmid pSpnP1 from a multidrug-resistant clone of Streptococcus pneumoniae.

A novel Streptococcus pneumoniae plasmid (pSpnP1; 5413bp) has been isolated from the multidrug-resistant clone Poland(23F)-16, and its complete nucleotide sequence has been determined. Sequence analysis predicted seven co-directional open reading frames and comparative analyses revealed that plasmid pSpnP1 is different to pDP1, the only previously described pneumococcal plasmid, whereas it is highly similar to pSt08, a plasmid from Streptococcus thermophilus. A double-stranded origin for replication similar to the replication origin of the pC194/pUB110 family was located upstream of the putative rep gene (orf2). It also contained a 144-bp region with over 60% identity to the single-stranded origin type A of the Streptococcus agalactiae plasmid pMV158/pLS1. Detection of single-stranded DNA by Southern blot analysis indicated that pSpnP1 replicates via a rolling circle mechanism. Interestingly, the product of orf1 has a putative Zonular occludens toxin conserved domain present in toxigenic strains of Vibrio cholerae. Real-time PCR assays revealed that this ORF was expressed. Hybridization experiments showed that the pSpnP1 replicon was unusual among other examined antibiotic-resistant pneumococcal clones, although the recombinant plasmids based on pSpnP1 were able to replicate in Bacillus subtilis and Lactococcus lactis.

Pneumococcus: the sugar-coated bacteria.

The study of Streptococcus pneumoniae (the pneumococcus) had been a central issue in medicine for many decades until the use of antibiotics became generalized. Many fundamental contributions to the history of microbiology should credit this bacterium: the capsular precipitin reaction, the major role this reaction plays in the development of immunology through the identification of polysaccharides as antigens, and, mainly, the demonstration, by genetic transformation, that genes are composed of DNA-the finding from the study of bacteria that has had the greatest impact on biology. Currently, pneumococcus is the most common etiologic agent in acute otitis media, sinusitis, and pneumonia requiring the hospitalization of adults. Moreover, meningitis is the leading cause of death among children in developing countries. Here I discuss the contributions that led to the explosion of knowledge about pneumococcus and also report some of the contributions of our group to the understanding of the molecular basis of three important virulence factors: lytic enzymes, pneumococcal phages, and the genes coding for capsular polysaccharides.

Pneumococcus: the sugarcoated bacteria / Pneumococcus: la bacteria recubierta de azúcar

The study of Streptococcus pneumoniae (the pneumococcus) had been a central issue in medicine for many decades until the use of antibiotics became generalized. Many fundamental contributions to the history of microbiology should credit this bacterium: the capsular precipitin reaction, the major role this reaction plays in the development of immunology through the identification of polysaccharides as antigens, and, mainly, the demonstration, by genetic transformation, that genes are composed of DNA—the finding from the study of bacteria that has had the greatest impact on biology. Currently, pneumococcus is the most common etiologic agent in acute otitis media, sinusitis, and pneumonia requiring the hospitalization of adults. Moreover, meningitis is the leading cause of death among children in developing countries. Here I discuss the contributions that led to the explosion of knowledge about pneumococcus and also report some of the contributions of our group to the understanding of the molecular basis of three important virulence factors: lytic enzymes, pneumococcal phages, and the genes coding for capsular polysaccharides (AU)

Hasta el empleo generalizado de los antibióticos, el estudio de Streptococcus pneumoniae (el neumococo) fue un tema central en medicina durante muchas décadas. Muchas contribuciones fundamentales de la historia de la microbiología se deben a esta bacteria: la reacción capsular de la precipitina, su destacado papel en el desarrollo de la inmunología mediante la identificación de polisacáridos como antígenos, y, principalmente, la demostración, por transformación genética, de que los genes están compuestos de DNA, que supuso el mayor impacto en biología a partir del estudio de las bacterias. Actualmente, el neumococo es el agente etiológico más frecuente en procesos de infección aguda del oído medio, sinusitis y neumonía que requieren hospitalización en adultos. Además, la meningitis es una de las principales causas de muerte en los niños de países en vías de desarrollo. Esta revisión trata las contribuciones que han llevado a un elevado nivel de conocimiento sobre el neumococo y describe algunas contribuciones de nuestro grupo al conocimiento de la base molecular de los tres factores principales de la virulencia, es decir, las enzimas líticas, los fagos y los genes que codifican los polisacáridos capsulares (AU)

Biofilm formation by Streptococcus pneumoniae: role of choline, extracellular DNA, and capsular polysaccharide in microbial accretion.

Streptococcus pneumoniae colonizes the human upper respiratory tract, and this asymptomatic colonization is known to precede pneumococcal disease. In this report, chemically defined and semisynthetic media were used to identify the initial steps of biofilm formation by pneumococcus during growth on abiotic surfaces such as polystyrene or glass. Unencapsulated pneumococci adhered to abiotic surfaces and formed a three-dimensional structure about 25 microm deep, as observed by confocal laser scanning microscopy and low-temperature scanning electron microscopy. Choline residues of cell wall teichoic acids were found to play a fundamental role in pneumococcal biofilm development. The role in biofilm formation of choline-binding proteins, which anchor to the teichoic acids of the cell envelope, was determined using unambiguously characterized mutants. The results showed that LytA amidase, LytC lysozyme, LytB glucosaminidase, CbpA adhesin, PcpA putative adhesin, and PspA (pneumococcal surface protein A) mutants had a decreased capacity to form biofilms, whereas no such reduction was observed in Pce phosphocholinesterase or CbpD putative amidase mutants. Moreover, encapsulated, clinical pneumococcal isolates were impaired in their capacity to form biofilms. In addition, a role for extracellular DNA and proteins in the establishment of S. pneumoniae biofilms was demonstrated. Taken together, these observations provide information on conditions that favor the sessile mode of growth by S. pneumoniae. The experimental approach described here should facilitate the study of bacterial genes that are required for biofilm formation. Those results, in turn, may provide insight into strategies to prevent pneumococcal colonization of its human host.

Characteristic signatures of the lytA gene provide a basis for rapid and reliable diagnosis of Streptococcus pneumoniae infections.

The nucleotide sequences of the lytA gene from 29 pneumococcal isolates of various serotypes and 22 additional streptococci of the mitis group (including two Streptococcus pseudopneumoniae strains) have been compared and found to correspond to 19 typical (927-bp-long) and 20 atypical (921-bp-long) alleles. All the Streptococcus pneumoniae strains harbored typical lytA alleles, whereas nonpneumococcal isolates belonging to the mitis group always carried atypical alleles. A sequence alignment showed that the main difference between typical and atypical lytA alleles resided in 102 nucleotide positions (including the 6 bp absent from atypical alleles). These nucleotides were perfectly conserved in all the typical alleles studied, and the corresponding nucleotides of the atypical alleles were also perfectly conserved. The presence in these signatures of distinctive restriction sites (namely, SnaBI, XmnI, and BsaAI) allowed the development of a simple, reliable, and fast method that combines PCR amplification of the lytA gene, digestion with BsaAI, and separation of the products by agarose gel electrophoresis. This assay allows the rapid and consistent identification of true S. pneumoniae strains and represents an improved diagnostic tool for the study of pneumococcal carriage.

Skl, a novel choline-binding N-acetylmuramoyl-L-alanine amidase of Streptococcus mitis SK137 containing a CHAP domain.

The skl gene from Streptococcus mitis SK137 encodes a peptidoglycan hydrolase (Skl) that has been purified and biochemically characterized. Analysis of the degradation products obtained by digestion of pneumococcal cell walls with Skl revealed that this enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), showing optimum activity at 30 degrees C and at a pH of 6.5. Skl is a unique member of the choline-binding family of proteins since it contains a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The CHAP domain of Skl showed homology to lysins of unknown especificity from a variety of streptococcal prophages. Skl represents the first characterized member of a new subfamily of CHAP-containing choline-binding proteins.

Allelic variation of polymorphic locus lytB, encoding a choline-binding protein, from streptococci of the mitis group.

The choline-binding protein LytB, an N-acetylglucosaminidase of Streptococcus pneumoniae, is the key enzyme for daughter cell separation and is believed to play a critical pathogenic role, facilitating bacterial spreading during infection. Because of these peculiarities LytB is a putative vaccine target. To determine the extent of LytB polymorphism, the lytB alleles from seven typical, clinical pneumococcal isolates of various serotypes and from 13 additional streptococci of the mitis group (12 atypical pneumococci and the Streptococcus mitis type strain) were sequenced. Sequence alignment showed that the main differences among alleles were differences in the number of repeats (range, 12 to 18) characteristic of choline-binding proteins. These differences were located in the region corresponding to repeats 11 to 17. Typical pneumococcal strains contained either 14, 16, or 18 repeats, whereas all of the atypical isolates except strains 1283 and 782 (which had 14 and 16 repeats, respectively) and the S. mitis type strain had only 12 repeats; atypical isolate 10546 turned out to be a DeltalytB mutant. We also found that there are two major types of alternating repeats in lytB, which encode 21 and 23 amino acids. Choline-binding proteins are linked to the choline-containing cell wall substrate through choline residues at the interface of two consecutive choline-binding repeats that create a choline-binding site. The observation that all strains contained an even number of repeats suggests that the duplication events that gave rise to the choline-binding repeats of LytB involved two repeats simultaneously, an observation that is in keeping with previous crystallographic data. Typical pneumococcal isolates usually grew as diplococci, indicating that an active LytB enzyme was present. In contrast, most atypical isolates formed long chains of cells that did not disperse after addition of purified LytB, suggesting that in these strains chains were produced through mechanisms unrelated to LytB.

Implications of physiological studies based on genomic sequences: Streptococcus pneumoniae TIGR4 synthesizes a functional LytC lysozyme.

The Streptococcus pneumoniae LytC lysozyme is responsible for autolysis at 30 degrees C (a temperature close to that of the upper respiratory tract), promotes DNA release in competent cultures, and participates in nasopharyngeal colonization. We show that the virulent pneumococcal TIGR4 strain encodes an active LytC enzyme, in contrast with genome-based predictions.

Ofloxacin-like antibiotics inhibit pneumococcal cell wall-degrading virulence factors.

The search for new drugs against Streptococcus pneumoniae (pneumococcus) is driven by the 1.5 million deaths it causes annually. Choline-binding proteins attach to the pneumococcal cell wall through domains that recognize choline moieties, and their involvement in pneumococcal virulence makes them potential targets for drug development. We have defined chemical criteria involved in the docking of small molecules from a three-dimensional structural library to the major pneumococcal autolysin (LytA) choline binding domain. These criteria were used to identify compounds that could interfere with the attachment of this protein to the cell wall, and several quinolones that fit this framework were found to inhibit the cell wall-degrading activity of LytA. Furthermore, these compounds produced similar effects on other enzymes with different catalytic activities but that contained a similar choline binding domain; that is, autolysin (LytC) and the phage lytic enzyme (Cpl-1). Finally, we resolved the crystal structure of the complex between the choline binding domain of LytA and ofloxacin at a resolution of 2.6 Angstroms. These data constitute an important launch pad from which effective drugs to combat pneumococcal infections can be developed.

Characterization of LytA-like N-acetylmuramoyl-L-alanine amidases from two new Streptococcus mitis bacteriophages provides insights into the properties of the major pneumococcal autolysin.

Two new temperate bacteriophages exhibiting a Myoviridae (phiB6) and a Siphoviridae (phiHER) morphology have been isolated from Streptococcus mitis strains B6 and HER 1055, respectively, and partially characterized. The lytic phage genes were overexpressed in Escherichia coli, and their encoded proteins were purified. The lytAHER and lytAB6 genes are very similar (87% identity) and appeared to belong to the group of the so-called typical LytA amidases (atypical LytA displays a characteristic two-amino-acid deletion signature). although they exhibited several differential biochemical properties with respect to the pneumococcal LytA, e.g., they were inhibited in vitro by sodium deoxycholate and showed a more acidic pH for optimal activity. However, and in sharp contrast with the pneumococcal LytA, a short dialysis of LytAHER or LytAB6 resulted in reversible deconversion to the low-activity state (E-form) of the fully active phage amidases (C-form). Comparison of the amino acid sequences of LytAHER and LytAB6 with that of the pneumococcal amidase suggested that Val317 might be responsible for at least some of the peculiar properties of S. mitis phage enzymes. Site-directed mutagenesis that changed Val317 in the pneumococcal LytA amidase to a Thr residue (characteristic of LytAB6 and LytAHER) produced a fully active pneumococcal enzyme that differs from the parental one only in that the mutant amidase can reversibly recover the low-activity E-form upon dialysis. This is the first report showing that a single amino acid residue is involved in the conversion process of the major S. pneumoniae autolysin. Our results also showed that some lysogenic S. mitis strains possess a lytA-like gene, something that was previously thought to be exclusive to Streptococcus pneumoniae. Moreover, the newly discovered phage lysins constitute a missing link between the typical and atypical pneumococcal amidases known previously.

Recent trends on the molecular biology of pneumococcal capsules, lytic enzymes, and bacteriophage.

Streptococcus pneumoniae has re-emerged as a major cause of morbidity and mortality throughout the world and its continuous increase in antimicrobial resistance is rapidly becoming a leading cause of concern for public health. This review is focussed on the analysis of recent insights on the study of capsular polysaccharide biosynthesis, and cell wall (murein) hydrolases, two fundamental pneumococcal virulence factors. Besides, we have also re-evaluated the molecular biology of the pneumococcal phage, their possible role in pathogenicity and in the shaping of natural populations of S. pneumoniae. Precise knowledge of the topics reviewed here should facilitate the rationale to move towards the design of alternative ways to combat pneumococcal disease.

Streptococcus pneumoniae and its bacteriophages: one long argument.

Infectious diseases currently kill more than 15 million people annually, and the WHO estimates that every year 1.6 million people die from pneumococcal diseases. Streptococcus pneumoniae (pneumococcus), a bacterium with a long biological pedigree, best illustrates the rapid evolution of antibiotic resistance, which has led to major public health concern. This article discusses the molecular basis of the two main virulence factors of pneumococcus, the capsule and cell-wall hydrolases, as well as new approaches to developing medicinal weapons for preventing pneumococcal infections. In addition, current knowledge regarding pneumococcal phages as potential contributors to virulence and the use of lytic enzymes encoded by these phages as therapeutic tools is reviewed.

Streptococcus pneumoniae and its bacteriophages: one long argument / Streptococcus pneumoniae y sus bacteriófagos: una prolongada controversia

Infectious diseases currently kill more than 15 million people annually, and the WHO estimates that every year 1.6 million people die from pneumococcal diseases. Streptococcus pneumoniae (pneumococcus), a bacterium with a long biological pedigree, best illustrates the rapid evolution of antibiotic resistance, which has led to major public health concern. This article discusses the molecular basis of the two main virulence factors of pneumococcus, the capsule and cell-wall hydrolases, as well as new approaches to developing medicinal weapons for preventing pneumococcal infections. In addition, current knowledge regarding pneumococcal phages as potential contributors to virulence and the use of lytic enzymes encoded by these phages as therapeutic tools is reviewed (AU)

Las enfermedades infecciosas matan anualmente a unos 15 millones de personas y la OMS estima que 1,6 millones de esas muertes se deben a infecciones neumocócicas. Streptococcus pneumoniae (neumococo), una bacteria con una notable contribución histórica a la biología, es el mejor ejemplo que ilustra el rápido desarrollo de la resistencia a los antibióticos, lo que puede originar un grave problema sanitario. Esta revisión analiza las bases moleculares de los dos factores principales de virulencia en el neumococo, la cápsula y las hidrolasas de la pared celular y describe nuevos enfoques para el desarrollo de nuevas herramientas médicas para prevenir las infecciones neumocócicas. También se analizan el conocimiento actual de la posible contribución de los fagos de neumococo a la virulencia de esta bacteria y el uso como arma terapéutica de las enzimas líticas codificadas por estos fagos (AU)

Peculiarities of the DNA of MM1, a temperate phage of Streptococcus pneumoniae.

The abundant presence of temperate phages in the chromosomes of clinical isolates of Streptococcus pneumoniae has been well documented. The genome of MM1, a temperate phage of pneumococcus, has been isolated as a DNA-protein complex. The protein is covalently bound to the DNA, was iodinated in vitro with Na125I, and has an Mr of 22,000. Electron microscopy and enzymatic analyses revealed that the MM1 genome is a linear, circularly permuted, terminally redundant collection of double-stranded DNA molecules packaged via a headful mechanism. The location of the pac site appears to be downstream of the terminase, between orf32 and orf34 of the MM1 genome.

Peculiarities of the DNA of MM1, a temperate phage of Streptococcus pneumoniae / Peculiaridades del DNA de MM1, un fago atemperado de Streptococcus pneumoniae

The abundant presence of temperate phages in the chromosomes of clinical isolates of Streptococcus pneumoniae has been well documented. The genome of MM1, a temperate phage of pneumococcus, has been isolated as a DNA-protein complex. The protein is covalently bound to the DNA, was iodinated in vitro with Na125I, and has an Mr of 22,000. Electron microscopy and enzymatic analyses revealed that the MM1 genome is a linear, circularly permuted, terminally redundant collection of double-stranded DNA molecules packaged via a heedful mechanism. The location of the pac site appears to be downstream of the terminase, between orf32 and orf34 of the MM1 genome (AU)

La abundante presencia de fagos atenuados en el cromosoma de aislados clínicos de Streptococcus pneumoniae ha sido bien documentada. El genoma de MM1, un fago atemperado de neumococo, se ha aislado como un complejo de DNA-proteína. La proteína unida covalentemente al DNA, fue yodada in vitro con Na125I, y tiene una Mr de 22.000. La microscopia electrónica y los análisis enzimáticos revelaron que el genoma de MM1 es una colección de moléculas lineales de DNA de doble cadena, permutadas circularmente y redundantes en la posición terminal, empaquetadas mediante un mecanismo de ensamblado de unidades genómicas discretas. También proponemos la localización del sitio pac en dirección 3´ de la terminas a, entreorf32 y orf34 del genoma de MM1 (AU)