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Rubens López

Publications and source records attributed to Rubens López.

At least 19 recordsLinked to original sources
PubMed2006

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.

Amidohydrolases↗
PubMed2006

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.

Amino Acid Sequence↗
PubMed2006

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.

Alleles↗
PubMed2006

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.

Bacterial Capsules↗
PubMed2005

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.

Anti-Bacterial Agents↗
PubMed2005

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.

Alleles↗
PubMed2005

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.

Base Sequence↗
PubMed2004

Genomic organization and molecular analysis of the inducible prophage EJ-1, a mosaic myovirus from an atypical pneumococcus.

We report the complete genomic sequence of EJ-1, an inducible prophage isolated from an atypical Streptococcus pneumoniae strain that belongs to the Myoviridae morphology family. The phage and bacterial recombinational sites (attachment sites) have been also determined. The genome of the EJ-1 prophage (42935 bp) is organized in 73 open reading frames (ORFs) and in at least five major clusters. Bioinformatic and N-terminal amino acid sequence analyses enabled the assignment of possible functions to 52 ORFs. The predicted proteins coded for the EJ-1 genome revealed similarities in the lysogeny, DNA replication, regulation, packaging, and head morphogenesis protein clusters with those from several siphoviruses infecting lactic acid bacteria. However, the proteins encoded by genes orf53 to orf64, corresponding to putative tail proteins of the virion, were very similar to those of the defective Bacillus subtilis myovirus PBSX with the notable exception of the gene product of orf56 (the tape measure tail protein) that was similar to proteins from phages infecting Gram-negative bacteria. The first description of the genome of a myovirus infecting a low G + C content Gram-positive bacterium, a member of a group embracing important human pathogens and industrial relevant species, will contribute to expand our current knowledge on phage biology and evolution.

Amino Acid Sequence↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

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.

DNA Packaging↗
PubMed2004

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.

Cell Wall↗
PubMed2003

Phage lytic enzymes as therapy for antibiotic-resistant Streptococcus pneumoniae infection in a murine sepsis model.

OBJECTIVES: Phage-coded lysins, i.e. murein hydrolases, are enzymes that destroy the cell wall of bacteria. A rapid killing of Streptococcus pneumoniae in the nasopharynx of mice has been described recently using a phage-coded murein hydrolase (enzybiotic). The in vivo effects of a dose-ranging treatment, using either of the phage-coded lytic enzymes Cpl-1 lysozyme or the Pal amidase, have been investigated here in a murine sepsis model. METHODS: Purified Pal amidase and/or Cpl-1 lysozyme were used alone or in combination. These enzymes were injected intraperitoneally at different times after challenge with 5 x 10(7) cfu of a type 6B, antibiotic-resistant S. pneumoniae clinical isolate. RESULTS: Animals challenged with 5 x 10(7) cfu of this strain alone died within 72 h, whereas a single intraperitoneal injection of Cpl-1 or Pal (200 microg; 1100 U) administered 1 h after the bacterial challenge was sufficient to effectively protect the mice, according to unpaired t-test (P<0.0001). Bacteraemia in unprotected mice reached colony counts >10(7) cfu/mL, whereas the mean colony count in lysin-protected animals was <10(6) cfu/mL over time and ultimately became undetectable. Interestingly, a synergic effect in vivo was observed with the combined use of 2.5 microg each of Cpl-1 and Pal. CONCLUSIONS: Our findings suggest strongly that phage lysins protect animals from bacteraemia and death. Moreover, the simultaneous attack of the pneumococcal peptidoglycan by a lysozyme and an amidase leads to a remarkable effect through enhanced destruction of the bacterial cell wall. The benefits of therapy with enzybiotics against pneumococcus reported here might warrant the examination of alternative strategies for the treatment of diseases caused by clinically relevant pathogens.

Amidohydrolases↗
PubMed2003

Molecular and biochemical analysis of the system regulating the lytic/lysogenic cycle in the pneumococcal temperate phage MM1.

The temperate phage MM1 forms stable lysogens in Streptococcus pneumoniae. We report here the first characterization of the lysogenic control region in Pneumococcus which contains two functional divergent promoters (P(R) and P(L)). MM1 encodes a 14-kDa cI protein (CI) that appears to be responsible for maintaining the lysogenic state in Pneumococcus since it prevents elongation of the transcripts controlled by P(R) and P(L).

Bacteriophages↗
PubMed2003

VO1, a temperate bacteriophage of the type 19A multiresistant epidemic 8249 strain of Streptococcus pneumoniae: analysis of variability of lytic and putative C5 methyltransferase genes.

A temperate bacteriophage (VO1) has been isolated from the Streptococcus pneumoniae type 19F multiresistant epidemic 8249 strain (South African strain). Structural analysis of the specific integration site, protein composition, restriction patterns, and molecular dissection of the lytic system of this phage revealed high sequence similarity with MM1, a temperate phage from the Spain23F-1 strain of pneumococcus, another multiresistant epidemic clone. The different pneumococcal strains sequenced so far exhibit an identical and single attB located in the same site of the genome. Remarkably, the LytA amidase coded by VO1 showed clear differences with that of the host bacterium in contrast with the situation previously documented for bacterial- and phage-coded amidases of pneumococcus. In addition, a new gene (orfmet) putatively coding for a C5 methyltransferase has been identified. A noticeable variability affecting the presence (or absence) of this supernumerary gene(s) in the same region of the genomes of three otherwise highly similar phages (i.e., VO1, MM1, and HB-3) suggests frequent recombinational events leading to introduce variability in this genome region. The peculiarities of genes like lytA and orfmet in VO1 provide interesting insights on mechanisms of horizontal transfer and lysogenic state co-evolution.

Amino Acid Sequence↗
PubMed2003

Genome organization and molecular analysis of the temperate bacteriophage MM1 of Streptococcus pneumoniae.

The genome of MM1 (40,248 bp), a temperate bacteriophage from the Spain(23F)-1 multiresistant epidemic clone of Streptococcus pneumoniae, is organized in 53 open reading frames (ORFs) and in at least five functional clusters. Bioinformatic and N-terminal amino acid sequence analyses enabled the assignment of possible functions to 26 ORFs. Analyses comparing the MM1 genome with those of other bacteriophages revealed similarities, mainly with genomes of phages infecting gram-positive bacteria, which suggest recent exchange of genes between species colonizing the same habitat.

Gene Order↗