Specificity of the autolysin of Streptococcus (Diplococcus) pneumoniae

A Streptococcus (Diplococcus) pneumoniae autolysin, partially purified from cellular autolysates, was optimally active at pH 7.0 and was stimulated by monovalent cations. Addition of autolysin to walls resulted in the appearance of only N-terminal l-alanine, whereas no glycosidase activity was observed. Walls which had been solubilized by autolysin were separated by gel filtration into a low-molecular-weight peptide containing amino acids in the same ratios found in intact walls and a high molecular fraction containing the amino acid-deficient peptidoglycan backbone. Thus, the major activity is an N-acetylmuramyl-l-alanine amidase. In addition, walls undergoing spontaneous lysis revealed no glycosidase activity but showed an increase in only N-terminal alanine. Autolysin, which was bound to walls in saline, was almost completely removed when walls were washed in distilled water, and all of the activity was recovered in the water wash fluid.     

Insertional inactivation of the major autolysin gene of Streptococcus pneumoniae.

The lytA gene encoding the major pneumococcal autolysin (N-acetylmuramoyl-L-alanine amidase) was inactivated by inserting the 2-kilobase MspI fragment of pE194 containing the staphylococcal ermC gene. Stable autolysis-deficient (Lyt-) mutants and their isogenic Lyt+ parents were used in experiments designed to test possible physiological functions of the amidase. No autolysis could be induced in the mutants grown at 37 degrees C by deoxycholate, by incubation in stationary phase, or by treatment with penicillin. On the other hand, the Lyt- mutants exhibited normal growth rates and yields and normal adaptive responses during shifts from one growth temperature or nutritional condition to another. There was no evidence for impeded cell separation (chain formation). Colonies of Lyt- insertional mutants produced normal hemolytic zones on blood agar; they showed normal (high) levels of competence for genetic transformation. Lyt- mutants were also able to produce type 3 and 6 capsular polysaccharides, and such strains showed the same degree of virulence in mice as did the isogenic Lyt+ parent. The physiological function(s) of the amidase remains a puzzle.     

A rapid procedure to detect the autolysin phenotype in Streptococcus pneumoniae

Abstract A simple and rapid procedure to detect autolysin-defective mutants of Streptococcus pneumoniae has been developed. The autolysin gene ( lyt ) can be introduced into the appropriate receptor strain by genetic transformation and the transformants are readily detected on the surface of semisynthetic medium (C medium) plates by using a membrane filter. A pneumococcal autolysin mutation ( lyt -4) behaved as a low-efficiency marker in genetic transformation.     

LytA, major autolysin of Streptococcus pneumoniae, requires access to nascent peptidoglycan

The pneumococcal autolysin LytA is a virulence factor involved in autolysis as well as in fratricidal- and penicillin-induced lysis. In this study, we used biochemical and molecular biological approaches to elucidate which factors control the cytoplasmic translocation and lytic activation of LytA. We show that LytA is mainly localized intracellularly, as only a small fraction was found attached to the extracellular cell wall. By manipulating the extracellular concentration of LytA, we found that the cells were protected from lysis during exponential growth, but not in the stationary phase, and that a defined threshold concentration of extracellular LytA dictates the onset of autolysis. Stalling growth through nutrient depletion, or the specific arrest of cell wall synthesis, sensitized cells for LytA-mediated lysis. Inhibition of cell wall association via the choline binding domain of an exogenously added enzymatically inactive form of LytA revealed a potential substrate for the amidase domain within the cell wall where the formation of nascent peptidoglycan occurs.     

Teichoic acid-containing muropeptides from Streptococcus pneumoniae as substrates for the pneumococcal autolysin.

Pneumococcal cell walls in which the normal phosphorylcholine component of the wall teichoic acids is replaced with phosphorylethanolamine cannot absorb the homologous autolytic enzyme and are completely resistant to autolytic degradation (S. Giudicelli and A. Tomasz, J. Bacteriol. 158:1188-1190, 1984). We have now isolated and characterized soluble teichoic acid-containing muropeptides from such cell walls and tested them as substrates for the pneumococcal autolytic enzyme. Both choline- and ethanolamine-containing muropeptides were hydrolyzed to the same extent by the enzyme. Furthermore, free choline concentrations that totally inhibited the digestion of pneumococcal cell walls in vivo and in vitro were without effect when the soluble substrates were used.     

Role of the major pneumococcal autolysin in the atypical response of a clinical isolate of Streptococcus pneumoniae.

The autolytic enzyme (an N-acetylmuramyl-L-alanine amidase) of a clinical isolate, strain 101/87, which is classified as an atypical pneumococcus, has been studied for the first time. The lytA101 gene coding for this amidase (LYTA101) has been cloned, sequenced, and expressed in Escherichia coli. The LYTA101 amidase has been purified and shown to be similar to the main autolytic enzyme (LYTA) present in the wild-type strain of Streptococcus pneumoniae, although it exhibits a lower specific activity, a higher sensitivity to inhibition by free choline, and a modified thermosensitivity with respect to LYTA. Most important, in contrast with the LYTA amidase, the activity of the LYTA101 amidase was inhibited by sodium deoxycholate. This property is most probably responsible of the deoxycholate-insensitive phenotype shown by strain 101/87. Phenotypic curing of strain 101/87 by externally adding purified LYTA or LYTA101 amidase restored in this strain some typical characteristics of the wild-type strain of pneumococcus (e.g., formation of diplo cells and sensitization to lysis by sodium deoxycholate), although the amount of the LYTA101 amidase required to restore these properties was much higher than in the case of the LYTA amidase. Our results indicate that modifications in the primary structure or in the mechanisms that control the activity of cell wall lytic enzymes seem to be responsible for the characteristics exhibited by some strains of S. pneumoniae that have been classically misclassified and should be now considered atypical pneumococcal strains.     

Temperate bacteriophages of Streptococcus pneumoniae that contain protein covalently linked to the 5'' ends of their DNA.

We have characterized three temperate bacteriophages of pneumococcus (HB-3, HB-623, and HB-746). Although all the phages belong to the same family, the polypeptide composition of the virions and the DNA restriction endonuclease analysis of their DNAs revealed differences among the three phages. The genomes of these bacteriophages have been isolated as DNA-protein complexes. The protein is specifically associated with the two 5' termini of the DNA as shown by experiments carried out with exonucleases. The protein bound to the DNA in the three phages studied, iodinated in vitro with 125I, has a molecular weight of 23,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the complexes with chaotropic agents suggested that the protein is covalently bound to the 5' termini of the DNA. Comparative pulsed-field gel electrophoresis analysis and Southern hybridization of the SmaI restriction fragments of DNAs from one lysogenic bacteria and its parental strain revealed that the prophage genome was integrated in the host chromosome.     

Local and systemic immunity against Streptococcus pneumoniae: Humoral responses against a non-capsulated temperature-sensitive mutant

Abstract Temperature-sensitive mutants of Streptococcus pneumoniae were isolated after chemical mutagenesis. Intranasal immunization with temperature-sensitive mutant J/3 induced higher levels of circulating antibody than those obtained after immunization with the heat-killed parental wild type. Moreover, local immunization with mutant J/3 induced high levels of anti- S. pneumoniae IgG and IgA in the lower respiratory tract, whereas only moderate IgG (and no IgA) antibodies were detected in lung lavage fluids from mice immunized intranasally with the heat-killed strain.     

Sequence of the Streptococcus pneumoniae bacteriophage HB-3 amidase reveals high homology with the major host autolysin.

We have sequenced a DNA fragment containing the pneumococcal bacteriophage HB-3 hbl gene, which codes for the phage lytic amidase. A remarkable nucleotide similarity (87.1%) between the lytA gene, coding for the pneumococcal amidase, the major autolysin of Streptococcus pneumoniae, and the hbl gene was found. This similarity completely disappeared outside the open reading frames coding for both amidases. The hbl gene transformed amidase-deficient strains of S. pneumoniae to the wild-type phenotype, and Southern blotting experiments provided evidence for recombination between donor and recipient genes. A comprehensive evaluation of these and previous results on the peptidoglycan hydrolases of S. pneumoniae and its bacteriophages suggested that recombination mechanisms participate in the evolution of the genes coding for these enzymes.     

Major peptidoglycan transglycosylase activity in Streptococcus pneumoniae that is not a penicillin-binding protein

Abstract In this communication, results support the conclusion that succinate can repress hexose-catabolizing enzymes in Rhizobium sp. 32H1 in a manner similar to catabolite repression, such as that seen in Pseudomonas aeruginosa [9,27]. Enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways were only detected in cells cultured on hexoses and were not present in succinate-grown cells. Initial enzymes of fructose and mannitol metabolism were present in cells gorwn on fructose and mannitol, respectively. NADP-linked 6-phosphogluconate dehydrogenase was not detected in cell-free extracts regardless of the carbon source.     

Hic, a novel surface protein of Streptococcus pneumoniae that interferes with complement function

The important human pathogen Streptococcus pneumoniae was found to absorb factor H, an inhibitor of complement, from human plasma. We identified the gene encoding a novel surface protein, factor H-binding inhibitor of complement (Hic), in the pspC locus of type 3 pneumococci. Unlike PspC proteins in other serotypes, Hic is anchored to the cell wall by means of an LPXTG motif, and the overall sequence homology to various PspC proteins is low. However, the NH(2)-terminal region showed significant homology to the NH(2)-terminal region of several PspC proteins. A fragment of Hic, covering this homologous region, was expressed as a glutathione S-transferase (GST) fusion protein. GST:Hic(39-261) bound radiolabeled factor H and inhibited binding of factor H to pneumococci of different serotypes. Interaction kinetics between GST:Hic(39-261) and factor H were studied with surface plasmon resonance and showed a high affinity binding (K(A) = 5 x 10(7), K(D) = 2.3 x 10(-)(8)). Mutant pneumococci lacking Hic showed no absorption of factor H in human plasma and no binding of radiolabeled factor H, suggesting that Hic is responsible for factor H-binding in type 3 pneumococci. Factor H-dependent inhibition of the alternative pathway was not diminished by the presence of GST:Hic(39-261). In addition, an intrinsic inhibitory effect of Hic is suggested.     

The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence

Streptococcus pneumoniae colonizes the nasopharynx in up to 40% of healthy subjects, and is a leading cause of middle ear infections (otitis media), meningitis and pneumonia. Pneumococci adhere to glycosidic receptors on epithelial cells and to immobilized fibronectin, but the bacterial adhesins mediating these reactions are largely uncharacterized. In this report we describe a novel pneumococcal protein PavA, which binds fibronectin and is associated with pneumococcal adhesion and virulence. The pavA gene, present in 64 independent isolates of S. pneumoniae tested, encodes a 551 amino acid residue polypeptide with 67% identical amino acid sequence to Fbp54 protein in Streptococcus pyogenes. PavA localized to the pneumococcal cell outer surface, as demonstrated by immunoelectron microscopy, despite lack of conventional secretory or cell-surface anchorage signals within the primary sequence. Full-length recombinant PavA polypeptide bound to immobilized human fibronectin in preference to fluid-phase fibronectin, in a heparin-sensitive interaction, and blocked binding of wild-type pneumococcal cells to fibronectin. However, a C-terminally truncated PavA' polypeptide (362 aa residues) failed to bind fibronectin or block pneumococcal cell adhesion. Expression of pavA in Enterococcus faecalis JH2-2 conferred > sixfold increased cell adhesion levels to fibronectin over control JH2-2 cells. Isogenic mutants of S. pneumoniae, either abrogated in PavA expression or producing a 42 kDa C-terminally truncated protein, showed up to 50% reduced binding to immobilized fibronectin. Inactivation of pavA had no effects on growth rate, cell morphology, cell-surface physico-chemical properties, production of pneumolysin, autolysin, or surface proteins PspA and PsaA. Isogenic pavA mutants of encapsulated S. pneumoniae D39 were approximately 104-fold attenuated in virulence in the mouse sepsis model. These results provide evidence that PavA fibronectin-binding protein plays a direct role in the pathogenesis of pneumococcal infections.     

Mutants of Escherichia coli that do not contain 1,4-diaminobutane (putrescine) or spermidine.

Strains of Escherichia coli K12 have been constructed which do not contain any of the polyamines normally present in a wild type strain, namely, 1,4-diaminobutane (putrescine) and spermidine. This phenotype arises as a consequence of the assembly into these strains of deletion mutations in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). The polyamine-deficient strains grow indefinitely in the absence of polyamines but with a growth rate one-third of that found in the presence of polyamines. These strains can act as hosts for bacteriophages T4, T7, and f2, although the latter phage is poorly adsorbed; they can also maintain F' factors, ColE1 and P1 plasmids, and lysogeny by bacteriophage lambda. In contrast, the production of bacteriophage lambda in the absence of polyamines is strikingly decreased (greater than 99%) either after infection of a nonlysogen or after induction of a lysogen. A polyamine-deficient Hfr strain can transfer its chromosome to a recipient at a normal rate, but the number of recombinants observed in a cross is decreased approximately 300-fold. No such effect is observed when only the F- recipient strain in a cross is polyamine deficient.     

New alkaloid antibiotics that target the DNA topoisomerase I of Streptococcus pneumoniae

Streptococcus pneumoniae has two type II DNA-topoisomerases (DNA-gyrase and DNA topoisomerase IV) and a single type I enzyme (DNA-topoisomerase I, TopA), as demonstrated here. Although fluoroquinolones target type II enzymes, antibiotics efficiently targeting TopA have not yet been reported. Eighteen alkaloids (seven aporphine and 11 phenanthrenes) were semisynthesized from boldine and used to test inhibition both of TopA activity and of cell growth. Two phenanthrenes (seconeolitsine and N-methyl-seconeolitsine) effectively inhibited both TopA activity and cell growth at equivalent concentrations (～17 μM). Evidence for in vivo TopA targeting by seconeolitsine was provided by the protection of growth inhibition in a S. pneumoniae culture in which the enzyme was overproduced. Additionally, hypernegative supercoiling was observed in an internal plasmid after drug treatment. Furthermore, a model of pneumococcal TopA was made based on the crystal structure of Escherichia coli TopA. Docking calculations indicated strong interactions of the alkaloids with the nucleotide-binding site in the closed protein conformation, which correlated with their inhibitory effect. Finally, although seconeolitsine and N-methyl-seconeolitsine inhibited TopA and bacterial growth, they did not affect human cell viability. Therefore, these new alkaloids can be envisaged as new therapeutic candidates for the treatment of S. pneumoniae infections resistant to other antibiotics.     

Transformation of Streptococcus sanguis Challis with a plasmid of Streptococcus pneumoniae

Abstract The present work is concerned with plasmid transformation of Streptococcus sanguis strain Challis with derivatives of pDP1/pSMB1, the only plasmid found to occur naturally in Streptococcus pneumoniae . Two recombinant plasmids derived from the cryptic pSMB1 were used: pDP27 (4.5 kb) conferring resistance to chloramphenicol (Cm), and pDP28 (7.8 kb), a shuttle plasmid, conferring resistance to Cm in Escherichia coli , and resistance to erythromycin (Em) in pneumococcus. It could be shown that pSMB1 can replicate in S. sanguis ; in fact, Challis strain V288 was transformed to Cm-resistance and to Em-resistance by pDP27 and pDP28 respectively.
Shuttle plasmid pDP28 can transform S. sanguis both when isolated from pneumococcus and from E. coli , albeit with a different efficiency. The low frequency of transformation observed when pDP28 was isolated from E. coli DH1 ( recA ) was shown to be due to lack of multimeric forms of the plasmid in the DNA preparations obtained from this strain. When pDP28 was isolated from E. coli C600 (RecA⁺), multimeric forms were present, and transformations of S. sanguis was more efficiency Using pDP28 as vector in cloning experiments, where S. sanguis was the host of the recombinant DNA molecules, treatment of the vector with alkaline phosphatase inhibited the recovery of recombinant clones.     

Electrotransformation of Streptococcus pneumoniae

Conditions of electroporation to transform encapsulated strains of Streptococcus pneumoniae with plasmid DNA have been defined. For a heavily encapsulated strain, an electroporation solution of 10–20% (v/v) glycerol and 3.2 kV cm^-1 field strength, 1000 Ω resistance and 25 μF capacitance were optimal. For lightly encapsulated or non-encapsulated strains, optimal conditions were a sucrose-based electroporation solution and 12.5 kV cm^-1 field strength, 200 Ω resistance and 25 μF capacitance.     

Mutants of Diplococcus pneumoniae that Lack Deoxyribonucleases and Other Activities Possibly Pertinent to Genetic Transformation

Mutants of Diplococcus pneumoniae that lacked the two major deoxyribonucleases of the cell—one an endonuclease, the other an exonuclease preferentially active on native deoxyribonucleic acid (DNA)—were obtained. The development of a method for detecting mutant colonies, based on the binding of methyl green to DNA, facilitated isolation of the mutants. Neither enzyme was essential for growth of the cells, for repair of ultraviolet damage, or for any phase of DNA-mediated transformation. Residual deoxyribonuclease activity in the double mutant corresponded to an exonuclease, approximately one-fifth as active as the major exonuclease, that attacked native and denatured DNA equally well. This activity appeared to be associated with the DNA-polymerase enzyme. A mutant that apparently lacked a cell wall lytic enzyme was also fully transformable. A mutant strain that was four times more sensitive to ultraviolet light than the wild type also transformed normally. Recipient cells of this strain were deficient in the repair of ultraviolet-irradiated transforming DNA. Mutants were found which, unlike the wild type, integrated donor markers only with high efficiency, thereby indicating that a particular cellular component that is susceptible to loss by mutation, such as an enzyme, is responsible for low integration efficiency.     

Antibiotic-tolerant mutants of Streptococcus pneumoniae that are not deficient in autolytic activity.

Several mutants of Streptococcus pneumoniae were isolated that appeared tolerant, to varying extents, to the lytic and bactericidal effects of some antibiotics that inhibit peptidoglycan synthesis, but were not deficient in autolytic activity. The method used to select the mutants was based on the survival of tolerant mutants during treatment with either bacitracin, benzylpenicillin, D-cycloserine plus beta-chloro-D-alanine, or vancomycin. Most (60 to 80%) of the surviving isolates were found to be deficient in autolytic activity, and these were rejected. The smaller proportion that had wild-type sensitivity to deoxycholate-induced lysis was studied further with respect to tolerance to the other antibiotics used in the selection procedures. Two of these mutants (selected by treatment with benzylpenicillin) were tolerant to either benzylpenicillin or D-cycloserine plus beta-chloro-D-alanine, but were supersusceptible, in terms of initiation of lysis, to either bacitracin or vancomycin. The minimal inhibitory concentration values of several antibiotics for these two mutants were identical to those for the wild-type strain. Moreover, the interaction of radioactive benzylpenicillin with the penicillin-binding proteins, examined in whole organisms, also appeared the same as previously found for either wild-type or autolytic-deficient strains of S. pneumoniae.     

Characterization and functional analysis of atl, a novel gene encoding autolysin in Streptococcus suis

Streptococcus suis serotype 2 (S. suis 2) is an important swine and human pathogen responsible for septicemia and meningitis. A novel gene, designated atl and encoding a major autolysin of S. suis 2 virulent strain HA9801, was identified and characterized in this study. The Atl protein contains 1,025 amino acids with a predicted molecular mass of 113 kDa and has a conserved N-acetylmuramoyl-l-alanine amidase domain. Recombinant Atl was expressed in Escherichia coli, and its bacteriolytic and fibronectin-binding activities were confirmed by zymography and Western affinity blotting. Two bacteriolytic bands were shown in the sodium dodecyl sulfate extracts of HA9801, while both were absent from the atl inactivated mutant. Cell chains of the mutant strain became longer than that of the parental strain. In the autolysis assay, HA9801 decreased to 20% of the initial optical density (OD) value, while the mutant strain had almost no autolytic activity. The biofilm capacity of the atl mutant was reduced ～30% compared to the parental strain. In the zebrafish infection model, the 50% lethal dose of the mutant strain was increased up to 5-fold. Furthermore, the adherence to HEp-2 cells of the atl mutant was 50% less than that of the parental strain. Based on the functional analysis of the recombinant Atl and observed effects of atl inactivation on HA9801, we conclude that Atl is a major autolysin of HA9801. It takes part in cell autolysis, separation of daughter cells, biofilm formation, fibronectin-binding activity, cell adhesion, and pathogenesis of HA9801.     

Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin.

A Streptococcus faecalis genomic bank was obtained by partial digestion with MboI and cloning into the SalI restriction site of pTZ18R. Screening of about 60,000 Escherichia coli transformants for cell wall lysis activity was done by exposing recombinant colonies grown on medium containing lyophilized Micrococcus lysodeikticus cells to chloroform and toluene vapors in order to release proteins. Because this procedure provoked cell death, colonies could not be used directly for transformant recovery; however, recovery was achieved by partial purification of plasmid DNA from active colonies on the agar plate and transformation of E. coli competent cells. About 60 recombinants were found. One of them (pSH6500) codes for a lytic enzyme active against S. faecalis and M. lysodeikticus cell walls. A shorter clone (pSH4000) was obtained by deleting an EcoRI fragment from the 6.5-kb original insert, leaving a 4-kb EcoRI-MboI insert; this subclone expressed the same lytic activity. Sequencing of a portion of pSH4000 revealed a unique open reading frame of 2,013 nucleotides coding for a 641-amino-acid (74-kDa) polypeptide and containing four 204-nucleotide direct repeats.