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PloS One Apr 2010Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections....
Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor alpha(2)-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.
Topics: Autolysis; Bacterial Proteins; Biofilms; DNA Transposable Elements; Extracellular Matrix Proteins; Gene Library; Genes, Bacterial; Mutation; Peptide Hydrolases; Polysaccharides; Staphylococcus aureus
PubMed: 20418950
DOI: 10.1371/journal.pone.0010146 -
Applied and Environmental Microbiology Jul 1999Baker's yeast suspensions having bacterial populations of 10(6) and 10(8) CFU/ml were subjected to autolysis processes designed to obtain yeast extracts (YE). The...
Baker's yeast suspensions having bacterial populations of 10(6) and 10(8) CFU/ml were subjected to autolysis processes designed to obtain yeast extracts (YE). The bacterial contaminants added to the yeast cell suspensions were produced with spent broths obtained from a commercial yeast production plant and contained 59% cocci (Leuconostoc, Aerococcus, Lactococcus) as well as 41% bacilli (Bacillus). Autolyses were conducted at four different pH levels (4.0, 5.5, 7.0, and 8.5) and with two autolysis-promoting agents (ethyl acetate and chitosan). Processing parameters were more important than the initial bacterial population in the development of contaminating bacteria during manufacture of YE. Drops in the viable bacterial population after a 24-h autolysis were observed when pH was adjusted to 4.0 or when ethyl acetate was added. A significant interaction was found between the effects of pH and autolysis promoters on the bacterial population in YE, indicating that the activity of ethyl acetate, as opposed to that of chitosan, was not influenced by pH.
Topics: Acetates; Bacillus; Chitin; Chitosan; Fermentation; Food Microbiology; Freezing; Hydrogen-Ion Concentration; Industrial Microbiology; Saccharomyces cerevisiae; Streptococcaceae
PubMed: 10388734
DOI: 10.1128/AEM.65.7.3261-3263.1999 -
Molecules and Cells Dec 2017More than 50% of sepsis cases are associated with pneumonia. Sepsis is caused by infiltration of bacteria into the blood via inflammation, which is triggered by the...
More than 50% of sepsis cases are associated with pneumonia. Sepsis is caused by infiltration of bacteria into the blood via inflammation, which is triggered by the release of cell wall components following lysis. However, the regulatory mechanism of lysis during infection is not well defined. Mice were infected with Streptococcus pneumoniae D39 wild-type (WT) and lipase mutant (ΔlipA) intranasally (pneumonia model) or intraperitoneally (sepsis model), and survival rate and pneumococcal colonization were determined. LipA and autolysin (LytA) levels were determined by qPCR and western blotting. S. pneumoniae Spd_1447 in the D39 (type 2) strain was identified as a lipase (LipA). In the sepsis model, but not in the pneumonia model, mice infected with the ΔlipA displayed higher mortality rates than did the D39 WT-infected mice. Treatment of pneumococci with serum induced LipA expression at both the mRNA and protein levels. In the presence of serum, the ΔlipA displayed faster lysis rates and higher LytA expression than the WT, both in vitro and in vivo. These results indicate that a pneumococcal lipase (LipA) represses autolysis via inhibition of LytA in a sepsis model.
Topics: A549 Cells; Animals; Autolysis; Bacterial Proteins; Blood Bactericidal Activity; Humans; Male; Mice; Mice, Inbred ICR; Pneumococcal Infections; Pneumonia, Pneumococcal; RAW 264.7 Cells; RNA, Messenger; Sepsis; Serum; Streptococcus pneumoniae; Virulence
PubMed: 29281779
DOI: 10.14348/molcells.2017.0201 -
Research in Microbiology Jan 1998Streptococcus pneumoniae is a pathogen in which the extracellular calcium concentration plays a major physiological role, in growth as well as in the induction of...
Streptococcus pneumoniae is a pathogen in which the extracellular calcium concentration plays a major physiological role, in growth as well as in the induction of competence for genetic transformation and activation of autolysis. Both responses are under the control of a protein activator exported in the medium. We have checked the impact of mutations which alter the regulation of competence and autolysis on experimental virulence. Isogenic encapsulated derivatives carrying the relevant mutations were serotype 3 smooth clones, obtained by transformation of the relevant rough strains with DNA from a serotype 3 smooth isolate. Survival kinetics and bacterial clearance from the blood were followed after intraperitoneal infection of Swiss mice with the different bacterial cultures. In this model, mutants showing an attenuation of virulence relative to the wild type fell into two classes. In the first, represented by the lytA::ery mutant V1095 defective for calcium-induced autolysis, attenuated virulence could be correlated with rapid bacterial clearance from the blood. In the second, represented by the dmb mutants V2200 and V3300, attenuation was associated with delayed bacterial clearance from the blood, and correlated with altered kinetics of calcium transport and of regulation of competence and autolysis. It appeared unlikely that attenuation of virulence for strains V2200 and V3300 was a direct consequence of their competence phenotype, since the com::ery mutants V1008 and V1019, defective for the production of the competence activator, were as virulent as the wild-type strain. Autolysis involving an N-acetyl-muramyl-alanine amidase encoded by lytA was also regulated by calcium. The inserted allele lytA0::ery further reduced virulence in the dmb1 background (V2200). This additive effect of lytA- to dmb1 points to different routes of virulence regulation by LYT and DMB1 and suggests that the kinetics of calcium traffic controls several pathways involved in the virulence of pneumococcus.
Topics: Animals; Autolysis; Bacteriolysis; Biological Transport; Calcium; Enzymes; Mice; Mutation; N-Acetylmuramoyl-L-alanine Amidase; Pneumococcal Infections; Streptococcus pneumoniae; Transformation, Bacterial; Virulence
PubMed: 9766204
DOI: 10.1016/s0923-2508(97)83618-2 -
Nature Communications Apr 2020Streptococcus pneumoniae serotype 1 is the predominant cause of invasive pneumococcal disease in sub-Saharan Africa, but the mechanism behind its increased invasiveness...
Streptococcus pneumoniae serotype 1 is the predominant cause of invasive pneumococcal disease in sub-Saharan Africa, but the mechanism behind its increased invasiveness is not well understood. Here, we use mouse models of lung infection to identify virulence factors associated with severe bacteraemic pneumonia during serotype-1 (ST217) infection. We use BALB/c mice, which are highly resistant to pneumococcal pneumonia when infected with other serotypes. However, we observe 100% mortality and high levels of bacteraemia within 24 hours when BALB/c mice are intranasally infected with ST217. Serotype 1 produces large quantities of pneumolysin, which is rapidly released due to high levels of bacterial autolysis. This leads to substantial levels of cellular cytotoxicity and breakdown of tight junctions between cells, allowing a route for rapid bacterial dissemination from the respiratory tract into the blood. Thus, our results offer an explanation for the increased invasiveness of serotype 1.
Topics: A549 Cells; Animals; Autolysis; Bacteremia; Bacterial Proteins; Bacterial Toxins; Cell Survival; Disease Models, Animal; Epithelial Cells; Female; Humans; Lung; Mice; Mice, Inbred BALB C; Nasopharynx; Pneumococcal Infections; Serogroup; Streptococcus pneumoniae; Streptolysins; Virulence; Virulence Factors
PubMed: 32312961
DOI: 10.1038/s41467-020-15751-6 -
British Journal of Cancer Sep 1973Normal and tumour tissues from rats, blood from normal and tumour bearing rats, and normal human blood were examined using the electron paramagnetic resonance (epr)...
Normal and tumour tissues from rats, blood from normal and tumour bearing rats, and normal human blood were examined using the electron paramagnetic resonance (epr) technique. At low temperature a triplet epr signal, which is known to be produced by a NO-haemoprotein complex, was detected in some tumour samples and in decaying normal liver. At room temperature all of the tumour samples examined gave a doublet signal. This signal was also detected in blood but not in other normal tissues. The signal has a g value of 2·0054 ± 0·0002 and a hyperfine splitting of 1·80 ± 0·05 G and is assigned to the ascorbyl free radical. Model experiments suggest that the appearance of detectable concentrations of this radical result from a disturbance of the normal state of the ascorbic acid, dehydroascorbic acid redox system. It was verified that cell division is not responsible for the ascorbyl radical although autolysis may be involved. A possible relationship between the formation of ascorbyl radicals and other paramagnetic species in tumours is discussed.
Topics: Animals; Ascorbic Acid; Autolysis; Blood Proteins; Carcinoma 256, Walker; Carcinoma, Hepatocellular; Cell Division; Electron Spin Resonance Spectroscopy; Liver Neoplasms; Lung Neoplasms; Methods; Models, Biological; Models, Chemical; Neoplasms; Nitric Oxide; Oxidation-Reduction; Rats; Sarcoma, Yoshida; Temperature
PubMed: 4355271
DOI: 10.1038/bjc.1973.145 -
Journal of Clinical Pathology Apr 1970Two methods of detecting early inapparent myocardial infarcts have been studied and their value in diagnostic practice compared. The better method proved to be the... (Comparative Study)
Comparative Study
Two methods of detecting early inapparent myocardial infarcts have been studied and their value in diagnostic practice compared. The better method proved to be the determination of the potassium to sodium ratio (ionic ratio) which falls in infarcted tissue within minutes of the onset of anoxia. The second method was nitro blue tetrazolium staining of gross sections of myocardium which revealed any infarct older than three and a half hours. As staining is dependent upon enzyme activity, the latter method is disturbed by autolysis. It was shown, on the other hand, that the ionic ratio (K(+)/Na(+)) was not affected by autolysis and was therefore well suited to forensic practice. Sixteen non-infarcted control hearts, plus the nine from cases of sudden death due to causes other than myocardial infarction, all yielded high ionic ratios (K(+)/Na(+)), average 1.4, and stained normally with tetrazolium (the normal controls). Positive control was provided by 20 histologically proven infarcts of which the ionic ratios (K(+)/Na(+)) were all low (average 0.7). Histochemical staining with tetrazolium delineated infarcted areas in each case. In a series of 29 sudden deaths, a cause of death other than myocardial infarction was found at necropsy in nine, mentioned above as normal controls. The remaining 20 hearts were not infarcted histologically, but were shown to be infarcted by examination of the ionic ratios (K(+)/Na(+)). These ratios were low (average 0.8) including three borderline ratios. Confirmatory evidence of infarction included nitro blue tetrazolium staining which revealed infarcts in 10 of the 20 cases, and clinical and necropsy observations. The ionic ratio (K(+)/Na(+)) decreases as the age of the infarct increases for at least 24 hours. Thereafter as healing proceeds, the ratio gradually reverts to normal. Thus, previous infarction and replacement fibrosis do not significantly alter the ionic ratio (K(+)/Na(+)). Nor is it changed by left ventricular hypertrophy, the presence of congestive cardiac failure, or digitalis therapy. It is suggested that macroscopic tetrazolium staining is a useful screening test for early inapparent myocardial infarcts. In cases where no infarct is delineated with that method estimation of the ionic ratio (K(+)/Na(+)) should be carried out on myocardium removed from standard areas on the anterior and posterior left ventricular walls.
Topics: Autolysis; Cardiomegaly; Death, Sudden; Digitalis Glycosides; Forensic Medicine; Heart Failure; Humans; Myocardial Infarction; Myocardium; Potassium; Sodium; Tetrazolium Salts
PubMed: 4246231
DOI: 10.1136/jcp.23.3.203 -
Journal of Applied Microbiology Nov 2000The autolysis of lactic acid bacteria plays a major role during cheese ripening. The aim of this study was to evaluate the autolytic properties and peptidoglycan... (Comparative Study)
Comparative Study
The autolysis of lactic acid bacteria plays a major role during cheese ripening. The aim of this study was to evaluate the autolytic properties and peptidoglycan hydrolase content of dairy leuconostocs. Autolysis of 59 strains of dairy Leuconostoc was examined under starvation conditions in potassium phosphate buffer. The ability of dairy leuconostocs to lyse is strain dependant and not related to the species. The peptidoglycan hydrolase profile of Leuc. mesenteroides subsp. mesenteroides 10L was analysed by renaturing gel electrophoresis. Two major activity bands migrating at 41 and 52 kDa were observed. According to the specificity analysis, strain 10L seems to contain a glycosidase and an N-acetyl-muramyl-L-alanine amidase, or an endopeptidase. The peptidoglycan hydrolase profiles of various Leuconostoc species were also compared. Several peptidoglycan hydrolase activities could be detected in the different Leuconostoc species. Further characterization of the peptidoglycan hydrolases will help to control autolysis of leuconostocs in cheese.
Topics: Bacteriolysis; Cheese; Electrophoresis, Polyacrylamide Gel; Food Microbiology; Hydrolases; Leuconostoc; Molecular Weight; Peptidoglycan
PubMed: 11119162
DOI: 10.1046/j.1365-2672.2000.01191.x -
PloS One 2016Soon after birth the mammalian gut microbiota forms a permanent and collectively highly resilient consortium. There is currently no robust method for re-deriving an...
Soon after birth the mammalian gut microbiota forms a permanent and collectively highly resilient consortium. There is currently no robust method for re-deriving an already microbially colonized individual again-germ-free. We previously developed the in vivo growth-incompetent E. coli K-12 strain HA107 that is auxotrophic for the peptidoglycan components D-alanine (D-Ala) and meso-diaminopimelic acid (Dap) and can be used to transiently associate germ-free animals with live bacteria, without permanent loss of germ-free status. Here we describe the translation of this experimental model from the laboratory-adapted E. coli K-12 prototype to the better gut-adapted commensal strain E. coli HS. In this genetic background it was necessary to complete the D-Ala auxotrophy phenotype by additional knockout of the hypothetical third alanine racemase metC. Cells of the resulting fully auxotrophic strain assembled a peptidoglycan cell wall of normal composition, as long as provided with D-Ala and Dap in the medium, but could not proliferate a single time after D-Ala/Dap removal. Yet, unsupplemented bacteria remained active and were able to complete their cell cycle with fully sustained motility until immediately before autolytic death. Also in vivo, the transiently colonizing bacteria retained their ability to stimulate a live-bacteria-specific intestinal Immunoglobulin (Ig)A response. Full D-Ala auxotrophy enabled rapid recovery to again-germ-free status. E. coli HS has emerged from human studies and genomic analyses as a paradigm of benign intestinal commensal E. coli strains. Its reversibly colonizing derivative may provide a versatile research tool for mucosal bacterial conditioning or compound delivery without permanent colonization.
Topics: Alanine; Alanine Racemase; Animals; Autolysis; Cell Wall; Diaminopimelic Acid; Escherichia coli K12; Gastrointestinal Microbiome; Gastrointestinal Tract; Germ-Free Life; Humans; Immunoglobulin A; Mice; Mice, Inbred C57BL; Microbial Consortia; Models, Animal; Peptidoglycan; Symbiosis
PubMed: 27002976
DOI: 10.1371/journal.pone.0151872 -
Current Biology : CB Jan 2016Extracellular DNA is an important component of the biofilm matrix. Now, Pseudomonas aeruginosa is shown to control autolysis through the production of HQNO, a...
Extracellular DNA is an important component of the biofilm matrix. Now, Pseudomonas aeruginosa is shown to control autolysis through the production of HQNO, a quorum-sensing-regulated respiratory poison. Thus, HQNO-driven autolysis links programmed cell death with quorum sensing and biofilm formation.
Topics: Humans; Biofilms; Pseudomonas aeruginosa; Quorum Sensing
PubMed: 26811896
DOI: 10.1016/j.cub.2015.12.018