scholarly journals Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation

2003 ◽  
Vol 185 (23) ◽  
pp. 6801-6808 ◽  
Author(s):  
Shannon A. Carroll ◽  
Torsten Hain ◽  
Ulrike Technow ◽  
Ayub Darji ◽  
Philippos Pashalidis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Separation ◽  
Bacterial Species ◽  
Surface Protein ◽  
Wild Type ◽  
Terminal Domain ◽  
Cell Wall Hydrolase ◽  
Characteristic Features ◽  
Type Gene

ABSTRACT A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes.

scholarly journals PASTA kinase-dependent control of peptidoglycan synthesis via ReoM is required for cell wall stress responses, cytosolic survival, and virulence in Listeria monocytogenes

2021 ◽  
Vol 17 (10) ◽  
pp. e1009881
Author(s):  
Jessica L. Kelliher ◽  
Caroline M. Grunenwald ◽  
Rhiannon R. Abrahams ◽  
McKenzie E. Daanen ◽  
Cassandra I. Lew ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Stress Responses ◽  
Pathogenic Bacteria ◽  
Critical Role ◽  
Wall Stress ◽  
Wild Type ◽  
Comprehensive Overview ◽  
Bacterial Physiology ◽  
Cell Wall Stress

Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen Listeria monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a β-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of reoM restores cell wall stress sensitivities and cytosolic survival defects of a ΔprkA mutant to nearly wild-type levels. While a ΔprkA mutant is defective for PG synthesis during cell wall stress, a double ΔreoM ΔprkA mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of reoM in a ΔprkA background almost fully restored virulence to wild-type levels. However, loss of reoM alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant Staphylococcus aureus. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence.

scholarly journals An Enterococcus faecium Secreted Antigen, SagA, Exhibits Broad-Spectrum Binding to Extracellular Matrix Proteins and Appears Essential for E. faecium Growth

2003 ◽  
Vol 71 (9) ◽  
pp. 5033-5041 ◽  
Author(s):  
Fang Teng ◽  
Magdalena Kawalec ◽  
George M. Weinstock ◽  
Waleria Hryniewicz ◽  
Barbara E. Murray
Keyword(s):  
Extracellular Matrix ◽  
Cell Wall ◽  
Broad Spectrum ◽  
Enterococcus Faecium ◽  
Collagen Type ◽  
Hydrolase Activity ◽  
Cell Wall Metabolism ◽  
Terminal Domain ◽  
Ecm Proteins ◽  
Cell Wall Hydrolase

ABSTRACT A gene encoding a major secreted antigen, SagA, was identified in Enterococcus faecium by screening an E. faecium genomic expression library with sera from patients with E. faecium-associated endocarditis. Recombinant SagA protein showed broad-spectrum binding to extracellular matrix (ECM) proteins, including fibrinogen, collagen type I, collagen type IV, fibronectin, and laminin. A fibrinogen-binding protein, purified from culture supernatants of an E. faecium clinical isolate, was found to match the N-terminal sequence of the predicted SagA protein and to react with the anti-SagA antibody, confirming that it was the SagA protein; this protein appeared as an 80- to 90-kDa smear on a Western blot that was sensitive to proteinase K and resistant to periodate treatment and glycoprotein staining. When overexpressed in E. faecium and Escherichia coli, the native and recombinant SagA proteins formed stable oligomers, apparently via their C-terminal domains. The SagA protein is composed of three domains: (i) a putative coiled-coil N-terminal domain that shows homology to the N-terminal domain of Streptococcus mutans SagA protein (42% similarity), previously shown to be involved in cell wall integrity and cell shape maintenance, and to the P45 protein of Listeria monocytogenes (41% similarity); (ii) a central domain containing direct repeats; and (iii) a C-terminal domain that is similar to that found in various proteins, including P45 (50% similarity) and P60 (52% similarity) of L. monocytogenes. The P45 and P60 proteins both have cell wall hydrolase activity, and the latter has also been shown to be involved in virulence, whereas cell wall hydrolase activity was not detected for SagA protein. The E. faecium sagA gene, like the S. mutans homologue, is located in a cluster of genes encoding proteins that appear to be involved in cell wall metabolism and could not be disrupted unless it was first transcomplemented, suggesting that the sagA gene is essential for E. faecium growth and may be involved in cell wall metabolism. In conclusion, the extracelluar E. faecium SagA protein is apparently essential for growth, shows broad-spectrum binding to ECM proteins, forms oligomers, and is antigenic during infection.

scholarly journals Aspergillus fumigatus RasA Regulates Asexual Development and Cell Wall Integrity

2008 ◽  
Vol 7 (9) ◽  
pp. 1530-1539 ◽  
Author(s):  
Jarrod R. Fortwendel ◽  
Kevin K. Fuller ◽  
Timothy J. Stephens ◽  
W. Clark Bacon ◽  
David S. Askew ◽  
...  
Keyword(s):  
Cell Wall ◽  
Hyphal Growth ◽  
Cell Wall Integrity ◽  
Signaling Cascades ◽  
Asexual Development ◽  
Wild Type ◽  
Calcofluor White ◽  
Ras Family ◽  
Type Gene ◽  
Increased Susceptibility

ABSTRACT The Ras family of proteins is a large group of monomeric GTPases. Members of the fungal Ras family act as molecular switches that transduce signals from the outside of the cell to signaling cascades inside the cell. A. fumigatus RasA is 94% identical to the essential RasA gene of Aspergillus nidulans and is the Ras family member sharing the highest identity to Ras homologs studied in many other fungi. In this study, we report that rasA is not essential in A. fumigatus, but its absence is associated with slowed germination and a severe defect in radial growth. The ΔrasA hyphae were more than two times the diameter of wild-type hyphae, and they displayed repeated changes in the axis of polarity during hyphal growth. The deformed hyphae accumulated numerous nuclei within each hyphal compartment. The ΔrasA mutant conidiated poorly, but this phenotype could be ameliorated by growth on osmotically stabilized media. The ΔrasA mutant also showed increased susceptibility to cell wall stressors, stained more intensely with calcofluor white, and was refractory to lysing enzymes used to make protoplasts, suggesting an alteration of the cell wall. All phenotypes associated with deletion of rasA could be corrected by reinsertion of the wild-type gene. These data demonstrate a crucial role for RasA in both hyphal growth and asexual development in A. fumigatus and provide evidence that RasA function is linked to cell wall integrity.

scholarly journals AsnB Mediates Amidation of Meso-Diaminopimelic Acid Residues in the Peptidoglycan of Listeria monocytogenes and Affects Bacterial Surface Properties and Host Cell Invasion

2021 ◽  
Vol 12 ◽  
Author(s):  
Lei Sun ◽  
Gil Rogiers ◽  
Pascal Courtin ◽  
Marie-Pierre Chapot-Chartier ◽  
Hélène Bierne ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Surface ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Diaminopimelic Acid ◽  
Bacterial Surface ◽  
Gram Positive Bacteria ◽  
First Time

A mutant of Listeria monocytogenes ScottA with a transposon in the 5' untranslated region of the asnB gene was identified to be hypersensitive to the antimicrobial t-cinnamaldehyde. Here, we report the functional characterization of AsnB in peptidoglycan (PG) modification and intracellular infection. While AsnB of Listeria is annotated as a glutamine-dependent asparagine synthase, sequence alignment showed that this protein is closely related to a subset of homologs that catalyze the amidation of meso-diaminopimelic acid (mDAP) residues in the peptidoglycan of other bacterial species. Structural analysis of peptidoglycan from an asnB mutant, compared to that of isogenic wild-type (WT) and complemented mutant strains, confirmed that AsnB mediates mDAP amidation in L. monocytogenes. Deficiency in mDAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the asnB mutant, including formation of shorter but thicker cells, susceptibility to lysozyme, loss of flagellation and motility, and a strong reduction in biofilm formation. In addition, the mutant showed reduced invasion of human epithelial JEG-3 and Caco-2 cells. Analysis by immunofluorescence microscopy revealed that asnB inactivation abrogated the proper display at the listerial surface of the invasion protein InlA, which normally gets cross-linked to mDAP via its LPXTG motif. Together, this work shows that AsnB of L. monocytogenes, like several of its homologs in related Gram-positive bacteria, mediates the amidation of mDAP residues in the peptidoglycan and, in this way, affects several cell wall and cell surface-related properties. It also for the first time implicates the amidation of peptidoglycan mDAP residues in cell wall anchoring of InlA and in bacterial virulence.

scholarly journals PASTA kinase-dependent control of peptidoglycan synthesis via ReoM is required for cell wall stress responses, cytosolic survival, and virulence in Listeria monocytogenes

2021 ◽  
Author(s):  
Jessica L. Kelliher ◽  
Caroline M. Grunenwald ◽  
Rhiannon R. Abrahams ◽  
McKenzie E. Daanen ◽  
Cassandra I. Lew ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Stress Responses ◽  
Pathogenic Bacteria ◽  
Critical Role ◽  
Wall Stress ◽  
Wild Type ◽  
Comprehensive Overview ◽  
Bacterial Physiology ◽  
Cell Wall Stress

Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen Listeria monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a β-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of reoM restores cell wall stress sensitivities and cytosolic survival defects of a ΔprkA mutant to nearly wild-type levels. While a ΔprkA mutant is defective for PG synthesis during cell wall stress, a double ΔreoM ΔprkA mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of reoM in a ΔprkA background almost fully restored virulence to wild-type levels. However, loss of reoM alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant Staphylococcus aureus. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence.

scholarly journals The atlA Operon of Streptococcus mutans: Role in Autolysin Maturation and Cell Surface Biogenesis

2006 ◽  
Vol 188 (19) ◽  
pp. 6877-6888 ◽  
Author(s):  
Sang-Joon Ahn ◽  
Robert A. Burne
Keyword(s):  
Cell Surface ◽  
Biofilm Formation ◽  
Cell Separation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Protein Profile ◽  
Critical Role ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Wild Type

ABSTRACT The Smu0630 protein (AtlA) was recently shown to be involved in cell separation, biofilm formation, and autolysis. Here, transcriptional studies revealed that atlA is part of a multigene operon under the control of at least three promoters. The morphology and biofilm-forming capacity of a nonpolar altA mutant could be restored to that of the wild-type strain by adding purified AtlA protein to the medium. A series of truncated derivatives of AtlA revealed that full activity required the C terminus and repeat regions. AtlA was cell associated and readily extractable from with sodium dodecyl sulfate. Of particular interest, the surface protein profile of AtlA-deficient strains was dramatically altered compared to the wild-type strain, as was the nature of the association of the multifunctional adhesin P1 with the cell wall. In addition, AtlA-deficient strains failed to develop competence as effectively as the parental strain. Mutation of thmA, which can be cotranscribed with atlA and encodes a putative pore-forming protein, resulted in a phenotype very similar to that of the AtlA-deficient strain. ThmA was also shown to be required for efficient processing of AtlA to its mature form, and treatment of the thmA mutant strain with full-length AtlA protein did not restore normal cell separation and biofilm formation. The effects of mutating other genes in the operon on cell division, biofilm formation, or AtlA biogenesis were not as profound. This study reveals that AtlA is a surface-associated protein that plays a critical role in the network connecting cell surface biogenesis, biofilm formation, genetic competence, and autolysis.

scholarly journals Listeria monocytogenes triggers noncanonical autophagy upon phagocytosis, but avoids subsequent growth-restricting xenophagy

2017 ◽  
Vol 115 (2) ◽  
pp. E210-E217 ◽  
Author(s):  
Gabriel Mitchell ◽  
Mandy I. Cheng ◽  
Chen Chen ◽  
Brittney N. Nguyen ◽  
Aaron T. Whiteley ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Bacterial Growth ◽  
Surface Protein ◽  
Time Lapse ◽  
Growth Restriction ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Subsequent Growth ◽  
Autophagic Process ◽  
And Function

Xenophagy is a selective macroautophagic process that protects the host cytosol by entrapping and delivering microbes to a degradative compartment. Both noncanonical autophagic pathways and xenophagy are activated by microbes during infection, but the relative importance and function of these distinct processes are not clear. In this study, we used bacterial and host mutants to dissect the contribution of autophagic processes responsible for bacterial growth restriction of Listeria monocytogenes. L. monocytogenes is a facultative intracellular pathogen that escapes from phagosomes, grows in the host cytosol, and avoids autophagy by expressing three determinants of pathogenesis: two secreted phospholipases C (PLCs; PlcA and PlcB) and a surface protein (ActA). We found that shortly after phagocytosis, wild-type (WT) L. monocytogenes escaped from a noncanonical autophagic process that targets damaged vacuoles. During this process, the autophagy marker LC3 localized to single-membrane phagosomes independently of the ULK complex, which is required for initiation of macroautophagy. However, growth restriction of bacteria lacking PlcA, PlcB, and ActA required FIP200 and TBK1, both involved in the engulfment of microbes by xenophagy. Time-lapse video microscopy revealed that deposition of LC3 on L. monocytogenes-containing vacuoles via noncanonical autophagy had no apparent role in restricting bacterial growth and that, upon access to the host cytosol, WT L. monocytogenes utilized PLCs and ActA to avoid subsequent xenophagy. In conclusion, although noncanonical autophagy targets phagosomes, xenophagy was required to restrict the growth of L. monocytogenes, an intracellular pathogen that damages the entry vacuole.

scholarly journals Candida albicans cell wall mannosidases, Dfg5 and Dcw1, regulate chitin synthesis

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Abhiram Maddi ◽  
Jaewon Kim ◽  
Harleen Sohi ◽  
Sujay Busarajan ◽  
Olga Glagovyak ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fluorescence Microscopy ◽  
Signaling Pathways ◽  
Cell Separation ◽  
Wild Type ◽  
Chitin Synthesis ◽  
Chitin Synthases ◽  
Conditional Mutant ◽  
Calcineurin Signaling

In Candida albicans chitin synthesis is important for cell wall integrity and may also have a role in emergence of drug-resistance. Our past studies showed that cell wall mannosidases, Dfg5 and Dcw1, regulate HOG MAPK signaling. In this study, we investigated how Dfg5 and Dcw1 regulate chitin synthesis by affecting HOG, PKC and Calcium-Calcineurin signaling pathways. DFG5 and DCW1 heterologous mutants (ES1 & ES195) and a conditional mutant (ES195+methionine/cysteine) were utilized. WT SC5314 served as negative control and Hog1 knock-out mutant as positive control. Fluorescence microscopy of calcofluor white (CFW) stained mutant and control strains was performed to observe chitin accumulation. Quantitative PCR analysis was performed to measure the relative expression of chitin synthases CHS1, CHS2, CHS3 and CHS8. Incubation with chitinase was done to determine cell separation using light microscopy and scanning electron microscopy (SEM) analysis. Fluorescence microscopy showed significantly increased chitin accumulation in the mutants as compared to wild type. Chitin accumulation was observed mainly at the budding sites indicating a cause for defective cell separation phenotype. Incubation with chitinase led to cell separation in the mutants. CHS2, CHS3 and CHS8 expression was observed to be significantly upregulated in the conditional mutant and HOG1 mutant as compared to the wild type. This upregulation was also observed when the cell wall integrity PKC pathway was activated. However, activation of the Calcium-calcineurin pathway downregulated chitin synthase expression in the mutants. Our data indicates that Dfg5 and Dcw1 regulate expression of chitin synthases via HOG MAPK, PKC and Calcium-calcineurin signaling pathways.

scholarly journals Spheroplast-mediated carbapenem tolerance in Gram-negative pathogens

2019 ◽  
Author(s):  
Trevor Cross ◽  
Brett Ransegnola ◽  
Jung-Ho Shin ◽  
Anna Weaver ◽  
Kathy Fauntleroy ◽  
...  
Keyword(s):  
Cell Wall ◽  
Treatment Failure ◽  
Bacterial Species ◽  
Enterobacter Cloacae ◽  
Wall Material ◽  
Klebsiella Aerogenes ◽  
Wild Type ◽  
Gram Negative ◽  
Clinically Significant ◽  
Carbapenem Antibiotic

AbstractAntibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied, yet likely widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae is able to tolerate exposure to the typically bactericidal β-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread tolerance is. Here, we have tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella aerogenes, but not Escherichia coli, exhibit moderate to high levels of tolerance to meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered to wild-type morphology and exponential growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.

scholarly journals Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative Pathogens

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Trevor Cross ◽  
Brett Ransegnola ◽  
Jung-Ho Shin ◽  
Anna Weaver ◽  
Kathy Fauntleroy ◽  
...  
Keyword(s):  
Cell Wall ◽  
Treatment Failure ◽  
Bacterial Species ◽  
Wall Material ◽  
Klebsiella Aerogenes ◽  
Wild Type ◽  
Gram Negative ◽  
Content Type ◽  
Clinically Significant ◽  
Carbapenem Antibiotic

ABSTRACT Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae can tolerate exposure to the typically bactericidal β-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread β-lactam tolerance is. Here, we tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae, but not Escherichia coli, exhibited moderate to high levels of tolerance of meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered wild-type morphology and growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.

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