A g-Type Lysozyme from Deep-Sea Hydrothermal Vent Shrimp Kills Selectively Gram-Negative Bacteria

Lysozyme is a key effector molecule of the innate immune system in both vertebrate and invertebrate. It is classified into six types, one of which is the goose-type (g-type). To date, no study on g-type lysozyme in crustacean has been documented. Here, we report the identification and characterization of a g-type lysozyme (named LysG1) from the shrimp inhabiting a deep-sea hydrothermal vent in Manus Basin. LysG1 possesses conserved structural features of g-type lysozymes. The recombinant LysG1 (rLysG1) exhibited no muramidase activity and killed selectively Gram-negative bacteria in a manner that depended on temperature, pH, and metal ions. rLysG1 bound target bacteria via interaction with bacterial cell wall components, notably lipopolysaccharide (LPS), and induced cellular membrane permeabilization, which eventually caused cell lysis. The endotoxin-binding capacity enabled rLysG1 to alleviate the inflammatory response induced by LPS. Mutation analysis showed that the bacterial binding and killing activities of rLysG1 required the integrity of the conserved α3 and 4 helixes of the protein. Together, these results provide the first insight into the activity and working mechanism of g-type lysozyme in crustacean and deep-sea organisms.

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG, while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end, with MpgA producing much longer peptidoglycan oligomers. We show that the cleavage site selectivity of MpgA is controlled by the LysM-like subdomain, which is required for its full functionality in cells. We propose that MltG’s ability to complement the loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

Identification and Functional Analysis of a Lysozyme Gene from Coridius Chinensis (Hemiptera: Dinidoridae)

Coridius chinensis is a valuable medicinal insect resource in China. Previous studies have indicated that the antibacterial and anticancer effects of the C. chinensis extract mainly come from the active polypeptides. Lysozyme is an effective immune effector in insect innate immunity and usually has excellent bactericidal effects. There are two kinds of lysozymes in insects, c-type and i-type, which play an important role in innate immunity and intestinal digestion. Studying lysozyme in C. chinensis will be helpful to further explore the evolutionary relationship and functional differences among lysozymes of various species and to determine whether they have biological activity and medicinal value. In this study, a lysozyme CcLys2 was identified from C. chinensis. CcLys2 contains 223 amino acid residues, and possesses a typical domain of the c-type lysozyme and a putative catalytic site formed by two conserved residues Glu32 and Asp50. Phylogenetic analysis showed that CcLys2 belongs to the H-branch of the c-type lysozyme. The analysis of spatiotemporal expression patterns indicated that CcLys2 was mainly expressed in the fat body of C. chinensis adults and was highly expressed in the second- and fifth-instar nymphs. In addition, CcLys2 was significantly up-regulated after injecting and feeding bacteria. In the bacterial inhibition assay, it was found that CcLys2 had antibacterial activity against Gram-positive bacteria at a low pH. These results indicate that CcLys2 has muramidase activity, involves in the innate immunity of C. chinensis, and is also closely related to the bacterial immune defense or digestive function of the intestine.

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

ABSTRACTThe peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. We are only beginning to understand the roles of peptidoglycan cleavage enzymes in cell wall assembly. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end. MpgA produces much longer peptidoglycan oligomers and we show that its cleavage site selectivity is controlled by the LysM-like subdomain, which is also present in MltG. We propose that MltG’s ability to complement loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

Molecular mechanism for self-protection against the type VI secretion system inVibrio cholerae

VgrG proteins form the spike of the type VI secretion system (T6SS) syringe-like complex. VgrG3 ofVibrio choleraedegrades the peptidoglycan cell wall of rival bacteriaviaits C-terminal region (VgrG3C) through its muramidase activity. VgrG3C consists of a peptidoglycan-binding domain (VgrG3CPGB) and a putative catalytic domain (VgrG3CCD), and its activity can be inhibited by its immunity protein partner TsiV3. Here, the crystal structure ofV. choleraeVgrG3CCDin complex with TsiV3 is presented at 2.3 Å resolution. VgrG3CCDadopts a chitosanase fold. A dimer of TsiV3 is bound in the deep active-site groove of VgrG3CCD, occluding substrate binding and distorting the conformation of the catalytic dyad. Gln91 and Arg92 of TsiV3 are located in the centre of the interface and are important for recognition of VgrG3C. Mutation of these residues destabilized the complex and abolished the inhibitory activity of TsiV3 against VgrG3C toxicity in cells. Disruption of TsiV3 dimerization also weakened the complex and impaired the inhibitory activity. These structural, biochemical and functional data define the molecular mechanism underlying the self-protection ofV. choleraeand expand the understanding of the role of T6SS in bacterial competition.

The Tape Measure Protein of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA35 Has an Active Muramidase Domain

ABSTRACTTailed double-stranded DNA (dsDNA) bacteriophages frequently harbor structural proteins displaying peptidoglycan hydrolytic activities. The tape measure protein fromStaphylococcus aureusbacteriophage vB_SauS-phiIPLA35 has a lysozyme-like and a peptidase_M23 domain. This report shows that the lysozyme-like domain (TG1) has muramidase activity and exhibitsin vitrolytic activity against liveS. aureuscells, an activity that could eventually find use in the treatment of infections.

Aggregates with lysozyme and ovalbumin show features of amyloid-like fibrils

The interaction of egg-white lysozyme with N-ovalbumin, the native form of egg-white ovalbumin with the denaturation temperature, Tm, of 78 °C, was investigated by the inhibition of lysozyme muramidase activity, differential scanning calorimetry, and circular dichroism assay as indicators. Signals for the interaction were the most prominent when the mixture of lysozyme and N-ovalbumin was co-heated at 72 °C, slightly lower than the Tm of N-ovalbumin. The interaction was also marked when unheated lysozyme was mixed with N-ovalbumin preheated at 72 °C. Moreover, the mixture rapidly formed fibrous precipitates, which were positive for thioflavin T fluorescent emission, a marker for the amyloid fibril formation. Also electron microscopic observation exhibited features of fibrils. The interaction potency of ovalbumin was ascribed to the tryptic fragment ILELPFASGT MSMLVLLPDE VSGLEQLESIINFEK (residues 229–263), derived from the 2B strands 2 and 3 of ovalbumin. From lysozyme, on the other hand, the chymotryptic peptide RNRCKGTDVQAW (residues 112–123), including cluster 6, and the chymotryptic/tryptic peptide GILQINSRW (residues 54–62), including cluster 3, were responsible for the interaction with N-ovalbumin. Interestingly, this nonamer peptide was found to have the ability to self-aggregate. To the authors knowledge, this may be the first report to document the possible involvement of dual proteins in the formation of amyloid-like fibrils.

The muramidase EtgA from enteropathogenic Escherichia coli is required for efficient type III secretion

Enteropathogenic Escherichia coli (EPEC) is an important cause of infectious diarrhoea. It colonizes human intestinal epithelial cells by delivering effector proteins into the host cell cytoplasm via a type III secretion system (T3SS) encoded within the chromosomal locus of enterocyte effacement (LEE). The LEE pathogenicity island also encodes a lytic transglycosylase (LT) homologue named EtgA. In the present work we investigated the significance of EtgA function in type III secretion (T3S). Purified recombinant EtgA was found to have peptidoglycan lytic activity in vitro. Consistent with this function, signal peptide processing and bacterial cell fractionation revealed that EtgA is a periplasmic protein. EtgA possesses the conserved glutamate characteristic of the LT family, and we show here that it is essential for enzymic activity. Overproduction of EtgA in EPEC inhibits bacterial growth and induces cell lysis unless the predicted catalytic glutamate is mutated. An etgA mutant is attenuated for T3S, red blood cell haemolysis and EspA filamentation. BfpH, a plasmid-encoded putative LT, was not able to functionally replace EtgA. Overall, our results indicate that the muramidase activity of EtgA is not critical but makes a significant contribution to the efficiency of the T3S process.