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The N -acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in nasopharyngeal colonization and is responsible for cell separation at the end of cell division; thus, Δ lytB mutants form long chains of cells. This paper reports the construction and properties of a defective pneumococcal mutant producing an inactive LytB protein (LytB E585A ). It is shown that an enzymatically active LytB is required for in vitro biofilm formation, as lytB mutants (either Δ lytB or producing the inactive LytB E585A ) are incapable of forming substantial biofilms, despite that extracellular DNA is present in the biofilm matrix. Adding small amounts (0.5 to 2.0 μg/ml) of exogenous LytB or some LytB constructs restored the biofilm-forming capacity of lytB mutants to wild-type levels. The LytB E585A mutant formed biofilm more rapidly than Δ lytB mutants in the presence of LytB. This suggests that the mutant protein acted in a structural role, likely through the formation of complexes with extracellular DNA. The chain-dispersing capacity of LytB allowed the separation of daughter cells, presumably facilitating the formation of microcolonies and, finally, of biofilms. A role for the possible involvement of LytB in the synthesis of the extracellular polysaccharide component of the biofilm matrix is also discussed. IMPORTANCE It has been previously accepted that biofilm formation in S. pneumoniae must be a multigenic trait because the mutation of a single gene has led to only to partial inhibition of biofilm production. In the present study, however, evidence that the N -acetylglucosaminidase LytB is crucial in biofilm formation is provided. Despite the presence of extracellular DNA, strains either deficient in LytB or producing a defective LytB enzyme formed only shallow biofilms., (Copyright © 2020 American Society for Microbiology.)

The cell wall glucosaminidase LytB of Streptococcus pneumoniae is a surface exposed protein involved in daughter cell separation, biofilm formation and contributes to different aspects of the pathogenesis process. In this study we have characterized the antibody responses after immunization of mice with LytB in the presence of alhydrogel as an adjuvant. Enzyme-linked immunosorbent assays measuring different subclasses of immunoglobulin G, demonstrated that the antibody responses to LytB were predominantly IgG1 and IgG2b, followed by IgG3 and IgG2a subclasses. Complement-mediated immunity against two different pneumococcal serotypes was investigated using sera from immunized mice. Immunization with LytB increased the recognition of S. pneumoniae by complement components C1q and C3b demonstrating that anti-LytB antibodies trigger activation of the classical pathway. Phagocytosis assays showed that serum containing antibodies to LytB stimulates neutrophil-mediated phagocytosis against S. pneumoniae. Animal models of infection including invasive pneumonia and sepsis were performed with two different clinical isolates. Vaccination with LytB increased bacterial clearance and induced protection demonstrating that LytB might be a good candidate to be considered in a future protein-based vaccine against S. pneumoniae., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Streptococcus pneumoniae (pneumococcus) is a major human pathogen. The main pneumococcal autolysin LytA and the pneumolysin Ply are two of the bacterium's most important virulence factors. The lytA- and ply-related genes are also found in other streptococci of the Mitis group (SMG). The precise characteristics of the lytA-related-but not the ply-related-genes of SMG and their prophages have been previously described. A search of the more than 400 SMG genomic sequences available in public databases (ca. 300 for S. pneumoniae), showed Streptococcus pseudopneumoniae IS7493 to harbor four ply-related genes, two of which (plyA and plyB) have 98% identical nucleotides. The plyA homolog of S. pseudopneumoniae is conserved in all S. pneumoniae strains, and seems to be included in a pathogenicity island together with the lytA gene. However, only nonencapsulated S. pneumoniae strains possess a plyB gene, which is part of an integrative and conjugative element. Notably, the existence of a bacterial lytA-related gene in a genome is linked to the presence of plyA and vice versa. The present analysis also shows there are eight main types of plyA-lytA genomic islands. A possible stepwise scenario for the evolution of the plyA-lytA island in S. pneumoniae is proposed., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The complement system is a key component of the host immune response for the recognition and clearance of Streptococcus pneumoniae. In this study, we demonstrate that the amidase LytA, the main pneumococcal autolysin, inhibits complement-mediated immunity independently of effects on pneumolysin by a complex process of impaired complement activation, increased binding of complement regulators, and direct degradation of complement C3. The use of human sera depleted of either C1q or factor B confirmed that LytA prevented activation of both the classical and alternative pathways, whereas pneumolysin inhibited only the classical pathway. LytA prevented binding of C1q and the acute-phase protein C-reactive protein to S. pneumoniae, thereby reducing activation of the classical pathway on the bacterial surface. In addition, LytA increased recruitment of the complement downregulators C4BP and factor H to the pneumococcal cell wall and directly cleaved C3b and iC3b to generate degradation products. As a consequence, C3b deposition and phagocytosis increased in the absence of LytA and were markedly enhanced for the lytA ply double mutant, confirming that a combination of LytA and Ply is essential for the establishment of pneumococcal pneumonia and sepsis in a murine model of infection. These data demonstrate that LytA has pleiotropic effects on complement activation, a finding which, in combination with the effects of pneumolysin on complement to assist with pneumococcal complement evasion, confirms a major role of both proteins for the full virulence of the microorganism during septicemia., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The emergence of Streptococcus pneumoniae strains displaying high levels of multidrug resistance is of great concern worldwide and a serious threat for the outcome of the infection. Modifications of the bacterial envelope by antibiotics may assist the recognition and clearance of the pathogen by the host immune system. Recognition of S. pneumoniae resistant strains by the complement component C3b was increased in the presence of specific anti-pneumococcal antibodies and subinhibitory concentrations of different macrolides and β-lactam antibiotics for all the strains investigated. However, C3b levels were unchanged in the presence of serum containing specific antibodies and sub-MICs of levofloxacin. To investigate whether LytA, the main cell wall hydrolase of S. pneumoniae, might be involved in this process, lytA-deficient mutants were constructed. In the presence of antibiotics, loss of LytA was not associated with enhanced C3b deposition on the pneumococcal surface, which confirms the importance of LytA in this interaction. The results of this study offer new insights into the development of novel therapeutic strategies using certain antibiotics by increasing the efficacy of the host immune response to efficiently recognize pneumococcal resistant strains.

Host- and phage-coded cell wall hydrolases have been used to fight Streptococcus pneumoniae growing as planktonic cells in vitro as well as in animal models. Until now, however, the usefulness of these enzymes in biofilm-grown pneumococci has gone untested. The antipneumococcal activity of different cell wall hydrolases produced by S. pneumoniae and a number of its phages was examined in an in vitro biofilm model. The major pneumococcal autolysin LytA, an N-acetylmuramoyl-l-alanine amidase, showed the greatest efficiency in disintegrating S. pneumoniae biofilms. The phage-encoded lysozymes Cpl-1 and Cpl-7 were also very efficient. Biofilms formed by the close pneumococcal relatives Streptococcus pseudopneumoniae and Streptococcus oralis were also destroyed by the phage endolysins but not by the S. pneumoniae autolysin LytA. A cooperative effect of LytA and Cpl-1 in the disintegration of S. pneumoniae biofilms was recorded.

Background: Streptococcus pneumoniae is a common colonizer of the human nasopharynx and one of the major pathogens causing invasive disease worldwide. Dissection of the molecular pathways responsible for colonization, invasion, and evasion of the immune system will provide new targets for antimicrobial or vaccine therapies for this common pathogen., Methodology/principal Findings: We have constructed mutants lacking the pneumococcal cell wall hydrolases (CWHs) LytB and LytC to investigate the role of these proteins in different phases of the pneumococcal pathogenesis. Our results show that LytB and LytC are involved in the attachment of S. pneumoniae to human nasopharyngeal cells both in vitro and in vivo. The interaction of both proteins with phagocytic cells demonstrated that LytB and LytC act in concert avoiding pneumococcal phagocytosis mediated by neutrophils and alveolar macrophages. Furthermore, C3b deposition was increased on the lytC mutant confirming that LytC is involved in complement evasion. As a result, the lytC mutant showed a reduced ability to successfully cause pneumococcal pneumonia and sepsis. Bacterial mutants lacking both LytB and LytC showed a dramatically impaired attachment to nasopharyngeal cells as well as a marked degree of attenuation in a mouse model of colonization. In addition, C3b deposition and phagocytosis was more efficient for the double lytB lytC mutant and its virulence was greatly impaired in both systemic and pulmonary models of infection., Conclusions/significance: This study confirms that the CWHs LytB and LytC of S. pneumoniae are essential virulence factors involved in the colonization of the nasopharynx and in the progress of invasive disease by avoiding host immunity.

Vancomycin is frequently added to standard therapy for pneumococcal meningitis. Although vancomycin-resistant Streptococcus pneumoniae strains have not been isolated, reports on the emergence of vancomycin-tolerant pneumococci are a cause of concern. To date, the molecular basis of vancomycin tolerance in S. pneumoniae is essentially unknown. We examined two vancomycin-tolerant clinical isolates, i.e. a purported autolysin negative (LytA(-)), serotype 23F isolate (strain S3) and the serotype 14 strain 'Tupelo', which is considered a paradigm of vancomycin tolerance. S3 was characterized here as carrying a frameshift mutation in the lytA gene encoding the main pneumococcal autolysin. The vancomycin tolerance of strain S3 was abolished by transformation to the autolysin-proficient phenotype. The original Tupelo strain was discovered to be a mixture: a strain showing a vancomycin-tolerant phenotype (Tupelo_VT) and a vancomycin-nontolerant strain (Tupelo_VNT). The two strains differed only in terms of a single mutation in the ciaH gene present in the VT strain. Most interestingly, although the vancomycin tolerance of Tupelo_VT could be overcome by increasing the LytA dosage upon transformation by a multicopy plasmid or by externally adding the autolysin, we show that vancomycin tolerance in S. pneumoniae requires the simultaneous presence of a mutated CiaH histidine kinase and capsular polysaccharide., (© 2010 Blackwell Publishing Ltd.)

According to a highly polymorphic region in the lytA gene, encoding the major autolysin of Streptococcus pneumoniae, two different families of alleles can be differentiated by PCR and restriction digestion. Here, we provide evidence that this polymorphic region arose from recombination events with homologous genes of pneumococcal temperate phages.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Very little is known about the in vivo regulation of the catalytic activity of the major pneumococcal autolysin (LytA), a surface-exposed enzyme that rules the self-destruction of pneumococcal cells through degradation of their peptidoglycan backbone. Two new crystal forms of the cell wall anchoring domain of LytA were obtained, and their structures were solved and refined to 2.4A and 2.8A resolution. The domain is a homodimer with a boomerang-like shape in which the tertiary structure of each monomer is comprised by six independent beta hairpins arranged in a superhelical fashion. Choline molecules at the hydrophobic interface of consecutive hairpins maintain this unique structure. The C-terminal hairpin (last 13 residues of LytA) in the solenoid is responsible for the formation of the catalytically active homodimer. Although the general fold in the structures derived from both crystal forms is essentially the same, two different conformations of the basic homodimer are observed. Biochemical approaches have demonstrated the fundamental role of the 11 C-terminal residues in the catalytic activity of LytA. The studies reported here reveal the importance of some amino acid residues at the C terminus in the determination of the relative distance of the active dimeric form of the autolysin, which appears to be essential for the catalytic activity of this enzyme.

The autolytic LytA amidase from 12 bile (deoxycholate)-insoluble streptococcal isolates (formerly classified as atypical Streptococcus pneumoniae) showing different antibiotic resistance patterns was studied. These atypical strains, which autolyze at the end of the stationary phase of growth, contain highly divergent lytA alleles (pairwise evolutionary distances of about 20%) compared to the lytA alleles of typical pneumococci. The atypical LytA amidases exhibit a peculiar deletion of two amino acids responsible for cell wall anchoring in the carboxy-terminal domain and have a reduced specific activity. These enzymes were inhibited by 1% deoxycholate but were activated by 1% Triton X-100, a detergent that could be used as an alternative diagnostic test for this kind of strain. Preparation of functional chimeric enzymes, PCR mutagenesis, and gene replacements demonstrated that the characteristic bile insolubility of these atypical strains was due to their peculiar carboxy-terminal domain and that the 2-amino-acid deletion was responsible for the inhibitory effect of deoxycholate. However, the deletion alone did not affect the specific activity of LytA. A detailed characterization of the genes encoding the 16S rRNA and SodA together with multilocus sequence typing indicated that the strains studied here are not a single clone and, although they cannot be strictly classified as typical pneumococci, they represent a quite diverse pool of organisms closely related to S. pneumoniae. The clinical importance of these findings is underlined by the role of the lytA gene in shaping the course of pneumococcal diseases. This study can also contribute to solving diagnostic problems and to understanding the evolution and pathogenic potential of species of the Streptococcus mitis group.