scholarly journals Anti-infectives in Drug Delivery—Overcoming the Gram-Negative Bacterial Cell Envelope

2016 ◽  
pp. 475-496 ◽  
Author(s):  
Florian Graef ◽  
Sarah Gordon ◽  
Claus-Michael Lehr
Keyword(s):  
Drug Delivery ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Gram Negative ◽  
Bacterial Cell Envelope

scholarly journals Cleavage of Braun’s lipoprotein Lpp from the bacterial peptidoglycan by a paralog of l,d-transpeptidases, LdtF

2021 ◽  
Vol 118 (19) ◽  
pp. e2101989118
Author(s):  
Raj Bahadur ◽  
Pavan Kumar Chodisetti ◽  
Manjula Reddy
Keyword(s):  
Bacterial Cell ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Hydrolytic Enzyme ◽  
Gram Negative ◽  
E Coli ◽  
Bacterial Cell Envelope ◽  
Cross Links ◽  
Bacterial Peptidoglycan ◽  
Elastic Polymer

The gram‐negative bacterial cell envelope is made up of an outer membrane (OM), an inner membrane (IM) that surrounds the cytoplasm, and a periplasmic space between the two membranes containing peptidoglycan (PG or murein). PG is an elastic polymer that forms a mesh-like sacculus around the IM, protecting cells from turgor and environmental stress conditions. In several bacteria, including Escherichia coli, the OM is tethered to PG by an abundant OM lipoprotein, Lpp (or Braun’s lipoprotein), that functions to maintain the structural and functional integrity of the cell envelope. Since its discovery, Lpp has been studied extensively, and although l,d-transpeptidases, the enzymes that catalyze the formation of PG−Lpp linkages, have been earlier identified, it is not known how these linkages are modulated. Here, using genetic and biochemical approaches, we show that LdtF (formerly yafK), a newly identified paralog of l,d-transpeptidases in E. coli, is a murein hydrolytic enzyme that catalyzes cleavage of Lpp from the PG sacculus. LdtF also exhibits glycine-specific carboxypeptidase activity on muropeptides containing a terminal glycine residue. LdtF was earlier presumed to be an l,d-transpeptidase; however, our results show that it is indeed an l,d-endopeptidase that hydrolyzes the products generated by the l,d-transpeptidases. To summarize, this study describes the discovery of a murein endopeptidase with a hitherto unknown catalytic specificity that removes the PG−Lpp cross-links, suggesting a role for LdtF in the regulation of PG–OM linkages to maintain the structural integrity of the bacterial cell envelope.

scholarly journals Cleavage of Braun lipoprotein Lpp from the bacterial peptidoglycan by a paralog of L,D-transpeptidases, LdtF

2021 ◽  
Author(s):  
Raj Bahadur ◽  
Pavan Kumar Chodisetti ◽  
Manjula Reddy
Keyword(s):  
Outer Membrane ◽  
Bacterial Cell ◽  
Drug Targets ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Hydrolytic Enzyme ◽  
Permeability Barrier ◽  
Gram Negative ◽  
E Coli ◽  
Bacterial Cell Envelope

AbstractGram-negative bacterial cell envelope is made up of an outer membrane (OM), an inner membrane (IM) that surrounds the cytoplasm, and a periplasmic space between the two membranes containing peptidoglycan (PG or murein). PG is an elastic polymer that forms a mesh-like sacculus around the IM protecting cells from turgor and environmental stress conditions. In several bacteria including E. coli, the OM is tethered to PG by an abundant OM lipoprotein, Lpp (or Braun lipoprotein) that functions to maintain the structural and functional integrity of the cell envelope. Since its discovery Lpp has been studied extensively and although L,D-transpeptidases, the enzymes that catalyse the formation of PG–Lpp linkages have been earlier identified, it is not known how these linkages are modulated. Here, using genetic and biochemical approaches, we show that LdtF (formerly yafK), a newly-identified paralog of L,D-transpeptidases in E. coli is a murein hydrolytic enzyme that catalyses cleavage of Lpp from the PG sacculus. LdtF also exhibits glycine-specific carboxypeptidase activity on muropeptides containing a terminal glycine residue. LdtF is earlier presumed to be an L,D-transpeptidase; however, our results show that it is indeed an L,D-endopeptidase that hydrolyses the products generated by the L,D-transpeptidases. To summarize, this study describes the discovery of a murein endopeptidase with a hitherto unknown catalytic specificity that removes the PG–Lpp cross-links suggesting a role for LdtF in regulation of PG-OM linkages to maintain the structural integrity of the bacterial cell envelope.Significance statementBacterial cell walls contain a unique protective exoskeleton, peptidoglycan, which is a target of several clinically important antimicrobials. In Gram-negative bacteria, peptidoglycan is covered by an additional lipid layer, outer membrane that serves as permeability barrier against entry of toxic molecules. In some bacteria, an extremely abundant lipoprotein, Lpp staples outer membrane to peptidoglycan to maintain the structural integrity of the cell envelope. In this study, we identify a previously unknown peptidoglycan hydrolytic enzyme that cleaves Lpp from the peptidoglycan sacculus and show how the outer membrane-peptidoglycan linkages are modulated in Escherichia coli. Overall, this study helps in understanding the fundamental bacterial cell wall biology and in identification of alternate drug targets for development of new antimicrobials.

scholarly journals Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores

2021 ◽  
Vol 17 (11) ◽  
pp. e1010051
Author(s):  
Dennis J. Doorduijn ◽  
Dani A. C. Heesterbeek ◽  
Maartje Ruyken ◽  
Carla J. C. de Haas ◽  
Daphne A. C. Stapels ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Membrane Attack Complex ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Complement Proteins ◽  
Bacterial Cell Envelope ◽  
Complement Resistance

Complement proteins can form membrane attack complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and ‘locked’ C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E. coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E. coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E. coli. By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

scholarly journals Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores

2021 ◽  
Author(s):  
Dennis J. Doorduijn ◽  
Dani A.C. Heesterbeek ◽  
Maartje Ruyken ◽  
Carla J.C. de Haas ◽  
Daphne A.C. Stapels ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Membrane Attack Complex ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Complement Proteins ◽  
Bacterial Cell Envelope

Complement proteins can form Membrane Attack Complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria, because a robust way to specifically prevent polymerization of C9 has been lacking. In this paper, polymerization of C9 was prevented without affecting the binding of C9 to C5b-8 by locking the first transmembrane helix domain of C9. We show that polymerization of C9 strongly enhanced bacterial cell envelope damage and killing by MAC pores for several Escherichia coli and Klebsiella strains. Moreover, we show that polymerization of C9 is impaired on complement-resistant E. coli strains that survive killing by MAC pores. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

scholarly journals The bacterial cell envelope as delimiter of anti-infective bioavailability – An in vitro permeation model of the Gram-negative bacterial inner membrane

2016 ◽  
Vol 243 ◽  
pp. 214-224 ◽  
Author(s):  
Florian Graef ◽  
Branko Vukosavljevic ◽  
Jean-Philippe Michel ◽  
Marius Wirth ◽  
Oliver Ries ◽  
...  
Keyword(s):  
Bacterial Cell ◽  
Cell Envelope ◽  
Inner Membrane ◽  
Gram Negative ◽  
Bacterial Cell Envelope ◽  
In Vitro Permeation

scholarly journals Expression of the Lipopolysaccharide-Modifying Enzymes PagP and PagL Modulates the Endotoxic Activity of Bordetella pertussis

2006 ◽  
Vol 74 (10) ◽  
pp. 5574-5585 ◽  
Author(s):  
Jeroen Geurtsen ◽  
Liana Steeghs ◽  
Hendrik-Jan Hamstra ◽  
Jan ten Hove ◽  
Alex de Haan ◽  
...  
Keyword(s):  
Pertussis Vaccine ◽  
Bordetella Pertussis ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Bacterial Cells ◽  
Gram Negative ◽  
Whole Cell ◽  
Bacterial Cell Envelope ◽  
Paradoxical Result ◽  
Endotoxic Activity

ABSTRACT Lipopolysaccharide (LPS) is one of the major constituents of the gram-negative bacterial cell envelope. Its endotoxic activity causes the relatively high reactogenicity of whole-cell vaccines. Several bacteria harbor LPS-modifying enzymes that modulate the endotoxic activity of the LPS. Here we evaluated whether two such enzymes, i.e., PagP and PagL, could be useful tools for the development of an improved and less reactogenic whole-cell pertussis vaccine. We showed that expression of PagP and PagL in Bordetella pertussis leads to increased and decreased endotoxic activity of the LPS, respectively. As expected, PagP activity also resulted in increased endotoxic activity of whole bacterial cells. However, more unexpectedly, this was also the case for PagL. This paradoxical result may be explained, in part, by an increased release of LPS, which we observed in the PagL-expressing cells.

scholarly journals How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

BioEssays ◽  
2019 ◽  
Vol 41 (10) ◽  
pp. 1900074 ◽  
Author(s):  
Dennis J. Doorduijn ◽  
Suzan H. M. Rooijakkers ◽  
Dani A. C. Heesterbeek
Keyword(s):  
Bacterial Cell ◽  
Cell Envelope ◽  
Membrane Attack Complex ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bacterial Cell Envelope

scholarly journals In Vitro Model of the Gram-Negative Bacterial Cell Envelope for Investigation of Anti-Infective Permeation Kinetics

2018 ◽  
Vol 4 (8) ◽  
pp. 1188-1196 ◽  
Author(s):  
Florian Graef ◽  
Robert Richter ◽  
Verena Fetz ◽  
Xabier Murgia ◽  
Chiara De Rossi ◽  
...  
Keyword(s):  
Bacterial Cell ◽  
In Vitro Model ◽  
Cell Envelope ◽  
Gram Negative ◽  
Bacterial Cell Envelope ◽  
Vitro Model ◽  
Permeation Kinetics
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