An analysis of LysM domain function in LytE when fulfilling the D,L-endopeptidase requirement for viability in Bacillus subtilis

The D,L-endopeptidase requirement states that Bacillus subtilis requires either the activity of the LytE or the CwlO enzyme for viability, therefore proving that these two enzymes can complement for each other despite their very different N-terminal domains. Here, we show that another D,L-endopeptidase, LytF, can also fulfill the D,L-endopeptidase requirement for viability, when expressed from the cwlO promoter. Both LytE and LytF contain N-terminally located LysM domains, three and five respectively. However, cells expressing another very similar D,L-endopeptidase CwlS, with four LysM domains were not viable. This led us to investigate whether a LytE protein with any one of its three LysM domains permuted can fulfill the D,L-endopeptidase requirement for viability. We found that the three LysM domains are not functionally equivalent and that the N-terminally located LysM domain plays a greater role for functioning of the LytE enzyme than the subsequent domains. Based on an investigation of orthologous enzymes in 19 B. subtilis species we propose an evolutionary model describing the development of the LytE-, CwlS- and LytF-type D,L-endopeptidases and their LysM domain repeats. In summary, these results show that the LytE enzyme has been optimized to fulfill the D,L-endopeptidase requirement for cell viability of B. subtilis with regard to the number and properties of LysM domains that mediate peptidoglycan-binding.

Improved antimicrobial spectrum of the N-acetylmuramoyl-L-alanine amidase from Latilactobacillus sakei upon LysM domain deletion

The gene encoding N-acetylmuramoyl-L-alanine amidase in Latilactobacillus sakei isolated from a fermented meat product was cloned in two forms: its complete sequence (AmiC) and without one of its anchoring LysM domains (AmiLysM4). Deletion of the LysM domain is believed to affect the target microorganism’s affinity to the cell wall, which influences antimicrobial activity. To compare activity and inhibitory spectra, AmiC and AmiLysM4 were expressed in Escherichia coli BL21. Using the zymography technique, two bands with lytic activity were observed, which were confirmed by LC-MS/MS analysis, with molecular masses of 71 kDa (AmiC) and 66 kDa (AmiLysM4). The recombinant proteins were active against Listeria innocua and Staphylococcus aureus strains; however, the inhibitory spectrum of AmiLysM4 was broader because AmiLysM4 could inhibit Leuconostoc mesenteroides and Weissella viridescens, which are microorganisms associated with food deterioration. Optimal temperature and pH values were determined for both proteins using L-alanine-p-nitroanilide hydrochloride as a substrate for N-acetylmuramoyl-L-alanine amidase activity. Both proteins showed similar maximum activity values for pH (8) and temperature (50°C). Furthermore, in silico predictions did not show differences for the catalytic region, but differences were found for the region called 3dom, which includes 3 of the 5 LysM domains. Therefore, the modification of the LysM domain offers new tools for the development of novel food biopreservatives.

Peptidoglycan Contribution to the B Cell Superantigen Activity of Staphylococcal Protein A

ABSTRACT Staphylococcus aureus causes reiterative and chronic persistent infections. This can be explained by the formidable ability of this pathogen to escape immune surveillance mechanisms. Cells of S. aureus display the abundant staphylococcal protein A (SpA). SpA binds to immunoglobulin (Ig) molecules and coats the bacterial surface to prevent phagocytic uptake. SpA also binds and cross-links variable heavy 3 (VH3) idiotype (IgM) B cell receptors, promoting B cell expansion and the secretion of nonspecific VH3-IgM via a mechanism requiring CD4+ T cell help. SpA binding to antibodies is mediated by the N-terminal Ig-binding domains (IgBDs). The so-called region X, uncharacterized LysM domain, and C-terminal LPXTG sorting signal for peptidoglycan attachment complete the linear structure of the protein. Here, we report that both the LysM domain and the LPXTG motif sorting signal are required for the B cell superantigen activity of SpA in a mouse model of infection. SpA molecules purified from staphylococcal cultures are sufficient to exert B cell superantigen activity and promote immunoglobulin secretion as long as they carry intact LysM and LPXTG motif domains with bound peptidoglycan fragments. The LysM domain binds the glycan chains of peptidoglycan fragments, whereas the LPXTG motif is covalently linked to wall peptides lacking glycan. These findings emphasize the complexity of SpA interactions with B cell receptors. IMPORTANCE The LysM domain is found in all kingdoms of life. While their function in mammals is not known, LysM domains of bacteria and their phage parasites are associated with enzymes that cleave or remodel peptidoglycan. Plants recognize microbe-associated molecular patterns such as chitin via receptors endowed with LysM-containing ectodomains. In plants, such receptors play equally important roles in defense and symbiosis signaling. SpA of S. aureus carries a LysM domain that binds glycan strands of peptidoglycan to influence defined B cell responses that divert pathogen-specific adaptive immune responses.

The Ncoa7 locus regulates V-ATPase formation and function, neurodevelopment and behaviour

Members of the Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic (TLDc) protein family are associated with multiple neurodevelopmental disorders, although their exact roles in disease remain unclear. For example, nuclear receptor coactivator 7 (NCOA7) has been associated with autism, although almost nothing is known regarding the mode-of-action of this TLDc protein in the nervous system. Here we investigated the molecular function of NCOA7 in neurons and generated a novel mouse model to determine the consequences of deleting this locus in vivo. We show that NCOA7 interacts with the cytoplasmic domain of the vacuolar (V)-ATPase in the brain and demonstrate that this protein is required for normal assembly and activity of this critical proton pump. Neurons lacking Ncoa7 exhibit altered development alongside defective lysosomal formation and function; accordingly, Ncoa7 deletion animals exhibited abnormal neuronal patterning defects and a reduced expression of lysosomal markers. Furthermore, behavioural assessment revealed anxiety and social defects in mice lacking Ncoa7. In summary, we demonstrate that NCOA7 is an important V-ATPase regulatory protein in the brain, modulating lysosomal function, neuronal connectivity and behaviour; thus our study reveals a molecular mechanism controlling endolysosomal homeostasis that is essential for neurodevelopment. Graphic abstract

A secreted LysM effector protects fungal hyphae through chitin-dependent homodimer polymerization

ABSTRACTPlants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases.

Genes coding for LysM domains in the dermatophyte Trichophyton rubrum: A transcription analysis

The filamentous fungus Trichophyton rubrum is a pathogen that causes superficial mycoses in humans, predominantly in keratinized tissues. The occurrence of dermatophytoses has increased in the last decades, mainly in immunocompromised patients, warranting research on the mechanisms involved in dermatophyte virulence. The genomes of dermatophytes are known to be enriched in genes coding for proteins containing the LysM domain, a carbohydrate-binding module, indicating the possible involvement of these genes in virulence. Although the LysM domains have already been described in other fungi, their biological functions in dermatophytes are unknown. Here we assessed the transcription of genes encoding proteins containing the LysM domains in T. rubrum grown on different substrates using quantitative real-time polymerase chain reaction. Some of these genes showed changes in transcription levels when T. rubrum was grown on keratin. In silico analyses suggest that some of these proteins share features, namely, they are anchored in the plasma membrane and contain the catalytic domain chitinase II and signal peptide domains. Here we show a detailed study of genes encoding the proteins with LysM-containing domains in T. rubrum, aiming to contribute to the understanding of their functions in dermatophytes.