-
Journal of Bacteriology Feb 2012Autolysis plays an essential role in bacterial cell division and lysis with β-lactam antibiotics. Accordingly, the expression of autolysins is tightly regulated by...
Autolysis plays an essential role in bacterial cell division and lysis with β-lactam antibiotics. Accordingly, the expression of autolysins is tightly regulated by several endogenous regulators, including ArlRS, a two component regulatory system that has been shown to negatively regulate autolysis in methicillin-sensitive Staphylococcus aureus (MSSA) strains. In this study, we found that inactivation of arlRS does not play a role in autolysis of methicillin-resistant S. aureus (MRSA) strains, such as community-acquired (CA)-MRSA strains USA300 and MW2 or the hospital-acquired (HA)-MRSA strain COL. This contrasts with MSSA strains, including Newman, SH1000, RN6390, and 8325-4, where autolysis is affected by ArlRS. We further demonstrated that the striking difference in the roles of arlRS between MSSA and MRSA strains is not due to the methicillin resistance determinant mecA. Among known autolysins and their regulators, we found that arlRS represses lytN, while no effect was seen on atl, lytM, and lytH expression in both CA- and HA-MRSA strains. Transcriptional-fusion assays showed that the agr transcripts, RNAII and RNAIII, were significantly more downregulated in the arlRS mutant of MW2 than the MSSA strain Newman. Importantly, provision of agr RNAIII in trans to the MW2 arlRS mutant via a multicopy plasmid induced autolysis in this MRSA strain. Also, the autolytic phenotype in the arlRS mutant of MSSA strain Newman could be rescued by a mutation in either atl or lytM. Together, these data showed that ArlRS impacts autolysis differently in MSSA and MRSA strains.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Bacteriolysis; Cloning, Molecular; Endopeptidases; Methicillin; Methicillin Resistance; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Mutation; N-Acetylmuramoyl-L-alanine Amidase; Penicillin-Binding Proteins; Plasmids; Protein Kinases; RNA, Bacterial; Trans-Activators
PubMed: 22139508
DOI: 10.1128/JB.06261-11 -
Cancer Cytopathology Apr 2021Although 10% formalin is a standard preservative in pancreatic FNAs, the effect of CytoLyt on pancreatic tissue preservation has not been systematically explored.
BACKGROUND
Although 10% formalin is a standard preservative in pancreatic FNAs, the effect of CytoLyt on pancreatic tissue preservation has not been systematically explored.
METHODS
Smears and cell blocks from CytoLyt-fixed (CF-CBs) and formalin-fixed (FF-CBs) pancreatic FNAs were blindly reviewed without knowledge of the fixative used, and the presence of tissue/tumor autolysis was noted. Controls included FF-CBs from pancreatic FNAs, CF-CBs from nonpancreatic FNAs, and 4 pancreatic FNAs with matched CF-CBs and FF-CBs.
RESULTS
We found that 62 of 85 (73%) pancreatic FNAs with CF-CBs showed significant autolysis, which was most pronounced in acinar cells and/or tumor cells with benign acinar cells in the background, compared with 2 of 46 (4%) FF-CBs (P < .0001) and 3 of 26 (12%) CF-CBs from nonpancreatic FNAs (73% vs 12%; P < .0001). Of the 4 pancreatic FNAs with matched CF-CBs and FF-CBs, all 4 CF-CBs showed marked autolysis versus none of the matched FF-CBs. Of the 23 (27%) pancreatic FNAs with CF-CBs that did not show autolysis, 10 had no acinar cells, and 7 had only minute tissue fragments on CB.
CONCLUSION
While CytoLyt is a useful fixative for nonpancreatic FNAs it is a suboptimal fixative for pancreatic FNAs and is associated with tissue/tumor autolysis in the majority of cases, influencing morphologic evaluation, and potentially immunocytochemical staining. Autolysis appears to be due to acinar enzymes whose effect is likely interrupted/inhibited by formalin fixation. Cytopathologists and cytotechnologists should be mindful of this pitfall and should avoid using CytoLyt as a fixative for pancreatic FNAs.
Topics: Acinar Cells; Autolysis; Biopsy, Fine-Needle; Female; Humans; Male; Pancreatic Neoplasms
PubMed: 33136337
DOI: 10.1002/cncy.22378 -
Frontiers in Microbiology 2019species are emerging as novel sources of antibiotics, but the regulation of their physiological metabolism is still poorly understood. In this work, we extracted AHL...
species are emerging as novel sources of antibiotics, but the regulation of their physiological metabolism is still poorly understood. In this work, we extracted AHL (acyl-homoserine lactone) autoinducers, identified the structures of AHLs and described the AHL quorum-sensing system in OH23. AHLs were isolated from the supernatant of OH23, and ESI-MS/MS (electrospray ionization mass spectrometry) analysis revealed biosynthesis of three different AHL chemical structures by OH23: -(3-oxohexanoyl)- homoserine lactone (HSL), 3-OH-C-HSL and C-HSL. The growth rate of AHL quorum-sensing knockout mutants was dramatically increased compared to that of wildtype. Sucrose consumptions were also twice as high in AHL quorum-sensing knockout mutants than that in wildtype in early-log phase. Additionally, expression of key genes related to sucrose metabolism α-glucosidase was enhanced in AHL quorum-sensing knockout mutants, which indicated that AHL quorum sensing negatively regulates sucrose uptake and metabolism which further affects the growth rate of . Furthermore, autolysis was strongly induced in AHL quorum-sensing knockout mutants compared to wildtype, suggesting that AHL quorum sensing plays a negative regulatory role in cell autolysis. Moreover, compared to wildtype, XSAC (-specific antibiotic compound) production was significantly increased in AHL knockout mutants in the early-log and late-log phases, and surface motility capabilities are also enhanced also in AHL knockout mutants; the normalized data of XSAC production and surface motility and expressions of key genes related to these two phenotypes reveal that growth rare and autolysis strongly affects XSAC biosynthesis and surface motility rather than AHL quorum-sensing system. Our results show that the AHL quorum-sensing system negatively regulates cell growth and autolysis, and further maintain nutrition homeostasis and population stability in .
PubMed: 31849892
DOI: 10.3389/fmicb.2019.02748 -
Journal of Oral Microbiology 2023Gingival crevicular fluid (GCF) constitutes the primary growth substrate for . The goal of this work was to evaluate the growth of different strains of on human serum...
AIMS
Gingival crevicular fluid (GCF) constitutes the primary growth substrate for . The goal of this work was to evaluate the growth of different strains of on human serum albumin (HSA), a major constituent of GCF.
METHODS
Growth of five different strains of in the HSA medium was examined and, surprisingly, three of the strains underwent autolysis within 24 h. Comparative transcriptomic analysis was used to identify genes involved in autolysis.
RESULTS
Two highly related reference strains (W50 and W83) differed dramatically in their survival when grown on HSA. Strain W83 grew fast and lysed within 24 h, while W50 survived for an additional 20 h. Differential gene expression analysis led us to a gene cluster containing enzymes involved in arginine metabolism and a gene predicted to be lytic murein transglycosylase, which are known to play a role in autolysis. Deletion of this gene (PG0139) resulted in a mutant that did not lyse, and complementation restored the HSA lysis phenotype, indicating that this enzyme plays a central role in the autolysis of .
CONCLUSIONS
undergoes autolysis when provided with HSA as a substrate for growth.
PubMed: 36570975
DOI: 10.1080/20002297.2022.2161182 -
Antimicrobial Agents and Chemotherapy Sep 2006The molecular basis of glycopeptide-intermediate S. aureus (GISA) isolates is not well defined though frequently involves phenotypes such as thickened cell walls and...
The molecular basis of glycopeptide-intermediate S. aureus (GISA) isolates is not well defined though frequently involves phenotypes such as thickened cell walls and decreased autolysis. We have exploited an isogenic pair of teicoplanin-susceptible (strain MRGR3) and teicoplanin-resistant (strain 14-4) methicillin-resistant S. aureus strains for detailed transcriptomic profiling and analysis of altered autolytic properties. Strain 14-4 displayed markedly deficient Triton X-100-triggered autolysis compared to its teicoplanin-susceptible parent, although microarray analysis paradoxically did not reveal significant reductions in expression levels of major autolytic genes atl, lytM, and lytN, except for sle1, which showed a slight decrease. The most important paradox was a more-than-twofold increase in expression of the cidABC operon in 14-4 compared to MRGR3, which was correlated with decreased expression of autolysis negative regulators lytSR and lrgAB. In contrast, the autolysis-deficient phenotype of 14-4 was correlated with both increased expression of negative autolysis regulators (arlRS, mgrA, and sarA) and decreased expression of positive regulators (agr RNAII and RNAIII). Quantitative bacteriolytic assays and zymographic analysis of concentrated culture supernatants showed a striking reduction in Atl-derived, extracellular bacteriolytic hydrolase activities in 14-4 compared to MRGR3. This observed difference was independent of the source of cell wall substrate (MRGR3 or 14-4) used for analysis. Collectively, our results suggest that altered autolytic properties in 14-4 are apparently not driven by significant changes in the transcription of key autolytic effectors. Instead, our analysis points to alternate regulatory mechanisms that impact autolysis effectors which may include changes in posttranscriptional processing or export.
Topics: Bacteriolysis; Cell Wall; Extracellular Fluid; Humans; Hydrolases; Methicillin Resistance; Octoxynol; Oligonucleotide Array Sequence Analysis; Peptide Hydrolases; Staphylococcus aureus; Teicoplanin; Transcription, Genetic
PubMed: 16940101
DOI: 10.1128/AAC.00113-06 -
Journal of Bacteriology Dec 2002Two distinctive colony morphologies were noted in a collection of Pseudomonas aeruginosa transposon insertion mutants. One set of mutants formed wrinkled colonies of...
Two distinctive colony morphologies were noted in a collection of Pseudomonas aeruginosa transposon insertion mutants. One set of mutants formed wrinkled colonies of autoaggregating cells. Suppressor analysis of a subset of these mutants showed that this was due to the action of the regulator WspR and linked this regulator (and the chemosensory pathway to which it belongs) to genes that encode a putative fimbrial adhesin required for biofilm formation. WspR homologs, related in part by a shared GGDEF domain, regulate cell surface factors, including aggregative fimbriae and exopolysaccharides, in diverse bacteria. The second set of distinctive insertion mutants formed colonies that lysed at their center. Strains with the most pronounced lysis overproduced the Pseudomonas quinolone signal (PQS), an extracellular signal that interacts with quorum sensing. Autolysis was suppressed by mutation of genes required for PQS biosynthesis, and in one suppressed mutant, autolysis was restored by addition of synthetic PQS. The mechanism of autolysis may involve activation of the endogenous prophage and phage-related pyocins in the genome of strain PAO1. The fact that PQS levels correlated with autolysis suggests a fine balance in natural populations of P. aeruginosa between survival of the many and persistence of the few.
Topics: Bacterial Adhesion; Bacterial Proteins; Bacteriolysis; Culture Media; DNA Transposable Elements; Gene Expression Regulation, Bacterial; Mutagenesis, Insertional; Mutation; Pseudomonas aeruginosa; Quinolones; Signal Transduction
PubMed: 12426335
DOI: 10.1128/JB.184.23.6481-6489.2002 -
Journal of Bacteriology Feb 1991Isogenic Tn551 mutants of a highly and uniformly methicillin-resistant strain of Staphylococcus aureus were tested for their rates of autolysis and cell wall degradation...
Isogenic Tn551 mutants of a highly and uniformly methicillin-resistant strain of Staphylococcus aureus were tested for their rates of autolysis and cell wall degradation in buffer and for cell wall turnover during growth. The normal (relatively fast) autolysis and turnover rates of the parent strain were retained in a Tn551 mutant in which the insert was located within the mec gene and which produced undetectable levels of penicillin-binding protein 2A. On the other hand, autolysis and cell wall turnover rates were greatly reduced in auxiliary mutants, i.e., mutants in which the transposon caused conversion of the high-level and uniform resistance of the parent strain to a variety of distinct heterogeneous expression types and greatly decreased resistance levels. All of these mutants contained an intact mec gene and produced normal amounts of penicillin-binding protein 2A, and one of the mutations was located in the femA region of the staphylococcal chromosome (B. Berger-Bachi, L. Barberis-Maino, A. Strassle, and F. H. Kayser, Mol. Gen. Genet. 219:263-269, 1989). Autolysis rates were related to the degree of residual methicillin resistance and to the sites of Tn551 insertion. Fast cell wall turnover may help expression of high-level methicillin resistance by providing a mechanism for the excision of abnormal (and potentially lethal) structural elements of the cell wall synthesized by the bacteria in the presence of methicillin.
Topics: Bacterial Proteins; Bacteriolysis; Carrier Proteins; Cell Wall; DNA Transposable Elements; Genes, Bacterial; Hexosyltransferases; Methicillin Resistance; Models, Biological; Muramoylpentapeptide Carboxypeptidase; Mutation; Penicillin-Binding Proteins; Penicillins; Peptidyl Transferases; Staphylococcus aureus
PubMed: 1846855
DOI: 10.1128/jb.173.3.1105-1110.1991 -
Bioscience Reports Nov 2020Calpain-1 and calpain-2 are highly structurally similar isoforms of calpain. The calpains, a family of intracellular cysteine proteases, cleave their substrates at... (Comparative Study)
Comparative Study
Calpain-1 and calpain-2 are highly structurally similar isoforms of calpain. The calpains, a family of intracellular cysteine proteases, cleave their substrates at specific sites, thus modifying their properties such as function or activity. These isoforms have long been considered to function in a redundant or complementary manner, as they are both ubiquitously expressed and activated in a Ca2+- dependent manner. However, studies using isoform-specific knockout and knockdown strategies revealed that each calpain species carries out specific functions in vivo. To understand the mechanisms that differentiate calpain-1 and calpain-2, we focused on the efficiency and longevity of each calpain species after activation. Using an in vitro proteolysis assay of troponin T in combination with mass spectrometry, we revealed distinctive aspects of each isoform. Proteolysis mediated by calpain-1 was more sustained, lasting as long as several hours, whereas proteolysis mediated by calpain-2 was quickly blunted. Calpain-1 and calpain-2 also differed from each other in their patterns of autolysis. Calpain-2-specific autolysis sites in its PC1 domain are not cleaved by calpain-1, but calpain-2 cuts calpain-1 at the corresponding position. Moreover, at least in vitro, calpain-1 and calpain-2 do not perform substrate proteolysis in a synergistic manner. On the contrary, calpain-1 activity is suppressed in the presence of calpain-2, possibly because it is cleaved by the latter protein. These results suggest that calpain-2 functions as a down-regulation of calpain-1, a mechanism that may be applicable to other calpain species as well.
Topics: Autolysis; Calpain; Enzyme Activation; Enzyme Stability; HCT116 Cells; HEK293 Cells; HeLa Cells; Humans; Proteolysis; Substrate Specificity; Time Factors; Troponin T
PubMed: 33078830
DOI: 10.1042/BSR20200552 -
The Journal of Biological Chemistry Jan 2016Proteolytic processing of human host cell factor 1 (HCF-1) to its mature form was recently shown, unexpectedly, to occur in a UDP-GlcNAc-dependent fashion within the...
Proteolytic processing of human host cell factor 1 (HCF-1) to its mature form was recently shown, unexpectedly, to occur in a UDP-GlcNAc-dependent fashion within the transferase active site of O-GlcNAc-transferase (OGT) (Lazarus, M. B., Jiang, J., Kapuria, V., Bhuiyan, T., Janetzko, J., Zandberg, W. F., Vocadlo, D. J., Herr, W., and Walker, S. (2013) Science 342, 1235-1239). An interesting mechanism involving formation and then intramolecular rearrangement of a covalent glycosyl ester adduct of the HCF-1 polypeptide was proposed to account for this unprecedented proteolytic activity. However, the key intermediate remained hypothetical. Here, using a model enzyme system for which the formation of a glycosyl ester within the enzyme active site has been shown unequivocally, we show that ester formation can indeed lead to proteolysis of the adjacent peptide bond, thereby providing substantive support for the mechanism of HCF-1 processing proposed.
Topics: Autolysis; Glutamates; Glycosylation; Protein Denaturation; Proteins; Proteolysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; beta-Glucosidase
PubMed: 26515062
DOI: 10.1074/jbc.C115.698696 -
Research in Microbiology Sep 2013Autolysis is an important process in cell wall turnover in Staphylococcus aureus, performed by several peptidoglycan hydrolases or so-called autolysins and controlled by...
Autolysis is an important process in cell wall turnover in Staphylococcus aureus, performed by several peptidoglycan hydrolases or so-called autolysins and controlled by many regulators. Rot is a global regulator that regulates numerous virulence genes, including genes encoding lipase, hemolysins, proteases and genes related to cell surface adhesion. The aim of our study was to determine whether Rot has the ability to regulate autolysis. We compared Triton-X-100-induced autolysis of S. aureus NCTC8325 and its rot knock-out mutant. We found that the rot mutant showed increased autolysis rates. By examining the transcript level of several autolysins and some known regulators responsible for regulating autolysis using real-time RT-PCR assays, we found that transcription of two autolysins (lytM, lytN) and one regulatory operon (lrgAB) was changed in the rot mutant. An in vitro approach was undertaken to determine which of these genes are directly controlled by Rot. Rot proteins were overproduced in Escherichia coli and purified. Gel mobility shift DNA binding assays were used and showed that in-vitro-purified Rot can directly bind to the promoter region of lytM, lytN, lrgA and lytS. We also tested biofilm formation of the rot mutant, and it showed enhancement in biofilm formation. Taken together, our results reveal that Rot affects autolysis by directly regulating autolysins LytM and LytN, and, via a regulatory system, LrgAB.
Topics: Bacterial Proteins; Bacteriolysis; Gene Expression Regulation, Bacterial; N-Acetylmuramoyl-L-alanine Amidase; Repressor Proteins; Staphylococcus aureus
PubMed: 23774059
DOI: 10.1016/j.resmic.2013.06.001