Targeting N-Acetylglucosaminidase in Staphylococcus aureus with Iminosugar Inhibitors.

Bacteria are capable of remarkable adaptations to their environment, including undesirable bacterial resistance to antibacterial agents. One of the most serious cases is an infection caused by multidrug-resistant Staphylococcus aureus, which has unfortunately also spread outside hospitals. Therefore, the development of new effective antibacterial agents is extremely important to solve the increasing problem of bacterial resistance. The bacteriolytic enzyme autolysin E (AtlE) is a promising new drug target as it plays a key role in the degradation of peptidoglycan in the bacterial cell wall. Consequently, disruption of function can have an immense impact on bacterial growth and survival. An in silico and in vitro evaluation of iminosugar derivatives as potent inhibitors of S. aureus (AtlE) was performed. Three promising hit compounds (1, 3 and 8) were identified as AtlE binders in the micromolar range as measured by surface plasmon resonance. The most potent compound among the SPR response curve hits was 1, with a KD of 19 µM. The KD value for compound 8 was 88 µM, while compound 3 had a KD value of 410 µM.

The characteristics of autolysins associated with cell separation in Bacillus subtilis.

The peptidoglycan hydrolases responsible for the cell separation of Bacillus subtilis cells are collectively referred to as autolysins. However, the role of each autolysin in the cell separation of B. subtilis is not fully understood. In this study, we constructed a series of cell separation-associated autolysin deficient strains and strains overexpressing the transcription factors SlrR and SinR, and the morphological changes of these strains in liquid culture were observed. The results showed that the absence of D,L-endopeptidases CwlS and LytF only increased the cell chain length in the early exponential phase. The absence of D,L-endopeptidase LytE or N-acetylmuramyl-L-alanine amidase LytC can cause cells to form chains throughout the growth of B. subtilis, although the cell chain length was significantly shortened during the stationary phase. However, the absence of peptidoglycan N-acetylglucosaminidase LytD only caused minor defect in cell separation. Therefore, we concluded that LytE and LytC were the major autolysins that ensure the timely separation of B. subtilis daughter cells, whereas CwlS, LytF, and LytD were the minor autolysins. In addition, overexpression of the transcription factors SinR and SlrR in the cwlS lytF lytC lytE mutant enabled B. subtilis cells to form ultra-long chains in the vegetative phase, and its biomass level was basically the same as that of the wild type. This led to the conclusion that besides inhibiting the expression of lytC and lytF, the SinR-SlrR complex also has other potential mechanisms to inhibit cell separation.IMPORTANCEIn this study, the effects of CwlS, LytC, LytD, LytF, LytE, and SinR-SlrR complex on the cell separation of Bacillus subtilis at different growth phases were studied, and an ultra-long-chained B. subtilis strain was constructed. In microbial fermentation, due to its large cell size, this ultra-long-chained B. subtilis strain may be more likely to be precipitated or intercepted during the removal of bacterial process with centrifugation and membrane filtration as the main methods, which is crucial to improve the purity of the product.

Endogenous cell wall degrading enzyme LytD is important for the biocontrol activity of Bacillus subtilis.

Autolysins are endogenous cell wall degrading enzymes (CWDEs) in bacteria that remodel the peptidoglycan layer of its own cell wall. In the Bacillus subtilis genome, at least 35 autolysin genes have been identified. However, the study of their roles in bacterial physiology has been hampered by their complexity and functional redundancy. B. subtilis GLB191 is an effective biocontrol strain against grape downy mildew disease, the biocontrol effect of which results from both direct effect against the pathogen and stimulation of the plant defense. In this study, we show that the autolysin N-acetylglucosaminidase LytD, a major autolysin of vegetative growth in B. subtilis, plays an important role in its biocontrol activity against grape downy mildew. Disruption of lytD resulted in reduced suppression of the pathogen Plasmopara viticola and stimulation of the plant defense. LytD is also shown to affect the biofilm formation and colonization of B. subtilis on grape leaves. This is the first report that demonstrates the role of an endogenous CWDE in suppressing plant disease infection of a biological control microorganism. These findings not only expand our knowledge on the biological function of autolysins but also provide a new target to promote the biocontrol activity of B. subtilis.

Monophosphoryl lipid A as a co-adjuvant in methicillin-resistant Staphylococcus aureus vaccine development: improvement of immune responses in a mouse model of infection.

To increase the effectiveness of methicillin-resistant Staphylococcus aureus vaccines (MRSA), a new generation of immune system stimulating adjuvants is necessary, along with other adjuvants. In some vaccines, monophosphoryl lipid A (MPLA) as a toll-like receptor 4 agonist is currently used as an adjuvant or co-adjuvant. MPLA could increase the immune response and vaccine immunogenicity. The current investigation assessed the immunogenicity and anti-MRSA efficacy of recombinant autolysin formulated in MPLA and Alum as co-adjuvant/adjuvant. r-Autolysin was expressed and purified by Ni-NTA affinity chromatography and characterized by SDS-PAGE. Then, the vaccine candidate formulation in MPLAs and Alum was prepared. To investigate the immunogenic responses, total IgG, isotype (IgG1 and IgG2a) levels, and cytokines (IL-4, IL-12, TNF-α, and IFN-γ) profiles were evaluated by ELISA. Also, the bacterial burden in internal organs, opsonophagocytosis, survival rate, and pathobiology changes was compared among the groups. Results demonstrated that mice immunized with the r-Autolysin + Alum + MPLA Synthetic and r-Autolysin + Alum + MPLA Biologic led to increased levels of opsonic antibodies, IgG1, IgG2a isotype as well as increased levels of cytokines profiles, as compared with other experimental groups. More importantly, mice immunized with MPLA and r-Autolysin exhibited a decrease in mortality and bacterial burden, as compared with the control group. The highest level of survival was seen in the r-Autolysin + Alum + MPLA Synthetic group. We concluded that both MPLA forms, synthetic and biological, are reliable candidates for immune response improvement against MRSA infection.

The Pneumococcal Protein SufC Binds to Host Plasminogen and Promotes Its Conversion into Plasmin.

Streptococcus pneumoniae causes otitis media, sinusitis, and serious diseases such as pneumonia and bacteremia. However, the in vivo dynamics of S. pneumoniae infections and disease severity are not fully understood. In this study, we investigated pneumococcal proteins detected in the bronchoalveolar lavage fluid of an S. pneumoniae-infected mouse, which were assumed to be expressed during infection. Analysis of three proteins with unknown infection-related functions revealed that recombinant Fe-S cluster assembly ATP-binding protein (SufC) binds to the host plasminogen and promotes its conversion into plasmin. SufC was detected in the bacterial cell-surface protein fraction, but it had no extracellular secretory signal. This study suggests that S. pneumoniae releases SufC extracellularly through LytA-dependent autolysis, binding to the bacterial cell surface and host plasminogen and promoting its conversion into plasmin. The recruitment of plasmin by S. pneumoniae is considered useful for bacterial survival and spread, and SufC is suggested to facilitate this process.

Novel P335-like Phage Resistance Arises from Deletion within Putative Autolysin yccB in Lactococcus lactis.

Lactococcus lactis and Lactococcus cremoris are broadly utilized as starter cultures for fermented dairy products and are inherently impacted by bacteriophage (phage) attacks in the industrial environment. Consequently, the generation of bacteriophage-insensitive mutants (BIMs) is a standard approach for addressing phage susceptibility in dairy starter strains. In this study, we characterized spontaneous BIMs of L. lactis DGCC12699 that gained resistance against homologous P335-like phages. Phage resistance was found to result from mutations in the YjdB domain of yccB, a putative autolysin gene. We further observed that alteration of a fused tail-associated lysin-receptor binding protein (Tal-RBP) in the phage restored infectivity on the yccB BIMs. Additional investigation found yccB homologs to be widespread in L. lactis and L. cremoris and that different yccB homologs are highly correlated with cell wall polysaccharide (CWPS) type/subtype. CWPS are known lactococcal phage receptors, and we found that truncation of a glycosyltransferase in the cwps operon also resulted in resistance to these P335-like phages. However, characterization of the CWPS mutant identified notable differences from the yccB mutants, suggesting the two resistance mechanisms are distinct. As phage resistance correlated with yccB mutation has not been previously described in L. lactis, this study offers insight into a novel gene involved in lactococcal phage sensitivity.

Design of a novel affinity probe using the cell wall-binding domain of a Listeria monocytogenes autolysin for pathogen detection.

IMPORTANCE: Human listeriosis is caused by consuming foods contaminated with the bacterial pathogen Listeria monocytogenes, leading to the development of a severe and life-threatening foodborne illness. Detection of L. monocytogenes present in food and food processing environments is crucial for preventing Listeria infection. The L. monocytogenes peptidoglycan hydrolase IspC anchors non-covalently to the bacterial surface through its C-terminal cell wall-binding domain (CWBD), CWBDIspC. This study explored the surface binding property of CWBDIspC to design, construct, characterize, and assess an affinity molecular probe for detecting L. monocytogenes. CWBDIspC recognized a cell wall ligand lipoteichoic acid that remains evenly displayed and mostly unoccupied on the bacterial surface for interaction with the exogenously added CWBDIspC. CWBDIspC, when fused to the enhanced green fluorescent protein reporter or covalently conjugated onto magnetic beads, exhibited the functionality as an antibody alternative for rapid detection and efficient separation of the pathogen.

Autolysin as a fibronectin receptor on the cell surface of Clostridium perfringens.

OBJECTIVE: Clostridium perfringens causes food poisoning and gas gangrene, a serious wound-associated infection. C. perfringens cells adhere to collagen via fibronectin (Fn). We investigated whether the peptidoglycan hydrolase of C. perfringens, i.e., autolysin (Acp), is implicated in Fn binding to C. perfringens cells. METHODS: This study used recombinant Acp fragments, human Fn and knockout mutants (C. perfringens 13 acp::erm and HN13 ΔfbpC ΔfbpD). Ligand blotting, Western blotting analysis, and complementation tests were performed. The Fn-binding activity of each mutant was evaluated by ELISA. RESULTS: From an Fn-binding assay using recombinant Acp fragments, Fn was found to bind to the catalytic domain of Acp. In mutant cells lacking Acp, Fn binding was significantly decreased, but was restored by the complementation of the acp gene. There are three known kinds of Fn-binding proteins in C. perfringens: FbpC, FbpD, and glyceraldehyde-3-phosphate dehydrogenase. We found no difference in Fn-binding activity between the mutant cells lacking both FbpC and FbpD (SAK3 cells) and the wild-type cells, indicating that these Fn-binding proteins are not involved in Fn binding to C. perfringens cells. CONCLUSIONS: We found that the Acp is an Fn-binding protein that acts as an Fn receptor on the surface of C. perfringens cells.

Autolysin Production from Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a model organism for various processes, from photosynthesis to cilia biogenesis, and a great chassis to learn more about biofuel production. This is due to the width of molecular tools available, which have recently expanded with the development of a modular cloning system but, most importantly, with CRISPR/Cas9 editing now being possible. This technique has proven to be more efficient in the absence of a cell wall by using specific mutants or by digesting Chlamydomonas cell wall using the mating-specific metalloprotease autolysin (also called gametolysin). Multiple protocols have been used and shared for autolysin production from Chlamydomonas cells; however, they provide very inconsistent results, which hinders the capacity to routinely perform CRISPR mutagenesis. Here, we propose a simple protocol for autolysin production requiring transfer of cells from plates into a dense liquid suspension, gametogenesis by overnight incubation before mixing of gametes, and enzyme harvesting after 2 h. This protocol has shown to be highly efficient for autolysin production regardless of precise control over cell density at any step. Requiring a minimal amount of labor, it will provide a simple, ready-to-go approach to produce an enzyme critical for the generation of targeted mutants.

Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against Staphylococcus aureus Strains.

Chimeric lysins composed of various combinations of cell wall-lysing (enzymatic) and cell-wall-binding (CWB) domains of endolysins, autolysins, and bacteriocins have been developed as alternatives to or adjuvants of conventional antibiotics. The screening of multiple chimeric lysin candidates for activity via E. coli expression is not cost effective, and we previously reported on a simple cell-free expression system as an alternative. In this study, we sufficiently improved upon this cell-free expression system for use in screening activity via a turbidity reduction test, which is more appropriate than a colony reduction test when applied in multiple screening. Using the improved protocol, we screened and compared the antibacterial activity of chimeric lysin candidates and verified the relatively strong activity associated with the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain of secretory antigen SsaA-like protein (ALS2). ALS2 expressed in E. coli showed two major bands, and the smaller one (subprotein) was shown to be expressed by an innate downstream promoter and start codon (ATG). The introduction of synonymous mutations in the promoter resulted in clearly reduced expression of the subprotein, whereas missense mutations in the start codon abolished antibacterial activity as well as subprotein production. Interestingly, most of the S. aureus strains responsible for bovine mastitis were susceptible to ALS2, but those from human and chicken were less susceptible. Thus, the simple and rapid screening method can be applied to select functional chimeric lysins and define mutations affecting antibacterial activity, and ALS2 may be useful in itself and as a lead molecule to control bovine mastitis.

Identification and characterization of a novel lytic peptidoglycan transglycosylase (MltC) in Shigella dysenteriae.

Shigellosis remains a worldwide health problem due to the lack of vaccines and the emergence of antibiotic-resistant strains. Shigella (S.) dysenteriae has rigid peptidoglycan (PG), and its tight regulation of biosynthesis and remodeling is essential for bacterial integrity. Lytic transglycosylases are highly conserved PG autolysins in bacteria that play essential roles in bacterial growth. However, their precise functions are obscure. We aimed to identify, clone, and express MltC, a unique autolysin in Escherichia (E.) coli C41 strain. The purification of recombinant MltC protein was performed using affinity chromatography and size-exclusion chromatography methods. The PG enzymatic activity of MltC was investigated using Zymogram and Fluorescein isothiocyanate (FITC)-labeled PG assays. Also, we aimed to detect its localization in bacterial fractions (cytoplasm and membrane) by western blot using specific polyclonal anti-MltC antibodies and its probable partners using immunoprecipitation and mass spectrometry applications. Purified MltC showed autolysin activity. Native MltC showed various locations in S. dysenteriae cells during different growth phases. In the Lag and early stationary phases, MltC was not found in cytoplasm and membrane fractions. However, it was detected in cytoplasm and membrane fractions during the exponential phase. In the late stationary phase, MltC was expressed in the membrane fraction only. Different candidate protein partners of MltC were identified that could be essential for bacterial growth and pathogenicity. This is the first study to suggest that MltC is indeed autolysin and could be a new drug target for the treatment of shigellosis by understanding its biological functions.

Glucomannan as a polysaccharide adjuvant improved immune responses against Staphylococcus aureus: Potency and efficacy studies.

Staphylococcus aureus is a gram-positive bacterium, representing one of the most important nosocomial pathogens. The treatment of infections, caused by S. aureus, has become increasingly intricate due to the emergence of highly resistant strains. Therefore, it is obvious that an effective prevention strategy against this bacterium could significantly decrease such infections. In the present study, the protective efficacy and immunological properties of recombinant autolysin, formulated in Montanide ISA266 and Alum adjuvants with Glucomannan as a polysaccharide, were assessed in the systemic mouse model of infection. Mice were immunized with the purified recombinant protein in various formulations in different groups and, subsequently, mice were challenged with 5 × 108 CFU of bacteria for the evaluation of their survival and bacterial clearances in the internal organs. ELISA was performed to determine the type of induced immunity, cytokine secretion (IFN-γ, IL-4, IL-2, and IL-17), and isotyping (IgG1 and IgG2a). In addition, we measured the opsonophagocytic activities of the antibodies. Results showed that immunization with r-autolysin + Alum + Glucomannan and r-autolysin + MontanideISA266+Glucomannan formulations significantly increased total IgG and isotypes (IgG1 and IgG2a), as compared with other vaccinated and control groups. Furthermore, the formulation of r-autolysin in Alum and MontanideISA266 adjuvants with Glucomannan enhanced IFN-γ, IL-4, and IL-17 cytokine secretion as well as protectivity, following experimental challenge. We concluded that Glucomannan has the potential to induce immune responses and would be used as an adjuvant factor in vaccine formulation.

Pneumococcal proteins ClpC and UvrC as novel host plasminogen binding factors.

Two plasminogen binding proteins were identified from a mouse infected with Streptococcus pneumoniae. The pneumococcal proteins were annotated as ATP-dependent Clp protease ATP-binding subunit (ClpC) and excinuclease ABC subunit C (UvrC) using the isobaric tags for relative and absolute quantification (iTRAQ) method. Recombinants of both proteins showed significant binding to plasminogen and were found to promote plasminogen activation by tissue-type plasminogen activator. In addition, ClpC and UvrC were LytA-dependently released into the culture supernatant and bound to the bacterial surface. These results suggest that S. pneumoniae releases ClpC and UvrC by autolysis and recruits them to the bacterial surface, where they bind to plasminogen and promote its activation, contributing to extracellular matrix degradation and tissue invasion.

Synthetic selenium nanoparticles as co-adjuvant improved immune responses against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital-acquired infections worldwide, which is resistant to many antibiotics, resulting in significant mortality in societies. Vaccination is a well-known approach to preventing disease. Autolysin, a surface-associated protein in S. aureus with multiple functions, is a suitable candidate for vaccine development. As a co-adjuvant, selenium nanoparticles (SeNPs) can increase the immune system, presumably resulting in increased vaccine efficacy. The present study evaluated the immunogenicity and defense of recombinant autolysin formulated in SeNPs and Alum adjuvants against MRSA. r-Autolysin was expressed and purified by the Ni-NTA affinity chromatography. SeNPs were synthetically obtained from sodium dioxide, followed by an assessment of shape and size using SEM and DLS. Balb/c mice were injected subcutaneously with 20 mg of r-autolysin formulated in Alum and SeNps adjuvants three times with the proper control group in 2 weeks intervals. Cytokine profile and isotyping ELISA were conducted to determine the type of induced immunity. Opsonophagocytosis tests assessed the functional activity of the vaccine, and the bacterial burden from the infected tissues was determined. Results showed that mice receiving SeNps and r-Autolysin had higher levels of total IgG and isotypes (IgG1 and IgG2a) and increased cytokine levels (IFN-γ, TNF-α, IL-12, and IL-4) as compared with those only receiving autolysin and PBS as a control. More importantly, mice immunized with SeNps and r-Autolysin exhibited a decrease in mortality and bacterial burden compared to the control group. We concluded that SeNps could stimulate immune responses and can be used as an adjuvant element in vaccine formulation.

Design and characterization of a novel lytic protein against Clostridium difficile.

Clostridium difficile (C. difficile) is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Autolysin is a lytic enzyme that hydrolyzes peptidoglycans of the bacterial cell wall, with a catalytic domain and cell wall-binding domains, proven to be involved in bacterial cell wall remodeling and cell division. Although autolysins in C. difficile have been reported, the autolysins have failed to yield impressive results when used as exogenous lytic agents. In this study, we expressed and characterized the binding domains (Cwp19-BD and Acd-BD) and catalytic domains (Cwp19-CD, Acd-CD, and Cwl-CD) of C. difficile autolysins, and the domains with the best binding specificity and lytic activity were selected towards C. difficile to design a novel lytic protein Cwl-CWB2. Cwl-CWB2 showed good biosafety with significantly low hemolysis and without cytotoxicity. The results of fluorescence analysis and lytic assay demonstrated that Cwl-CWB2 has higher binding specificity and stronger lytic activity with a minimum inhibitory concentration at 13.39 ± 5.80 µg/mL against living C. difficile cells, which is significantly stronger than commercial lysozyme (3333.33 ± 1443.37 µg/mL) and other reported C. difficile autolysins. Besides, Cwl-CWB2 exhibited good stability as about 75% of the lytic activity was still retained when incubated at 37 °C for 96 h, which is considered to be a potential antimicrobial agent to combat C. difficile. KEY POINTS: • Several binding domains and catalytic domains, deriving from several Clostridium difficile autolysins, were expressed, purified, and functionally characterized. • A novel C. difficile lytic protein Cwl-CWB2 was designed from C. difficile autolysins. • The binding specificity and lytic activity of Cwl-CWB2 against C. difficile showed advantages compared with other reported C. difficile autolysins. • Cwl-CWB2 exhibited significantly low hemolysis and cytotoxicity against normal-derived colon mucosa 460 cell.

Recombinant PBP2a/autolysin conjugate as PLGA-based nanovaccine induced humoral responses with opsonophagocytosis activity, and protection versus methicillin-resistant Staphylococcus aureus infection.

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) reasons extreme infections, can resist various conventional antimicrobial agents, and cause morbidity and mortality worldwide. Vaccination seems to help modulate MRSA infections. Nanovaccine is considered a novel strategy in vaccine technology. The primary purpose of the present study was to develop a conjugate vaccine based on recombinant PBP2a and MRSA autolysin formulated in PLGA as a nanoparticle capable of enhancing protective responses against MRSA in the murine model. Materials and Methods: Recombinant PBP2a and autolysin have been expressed and purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity column and characterized by SDS-PAGE and western blot. PLGA was bound to recombinant proteins by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and adipic acid dihydrazide (ADH) as a linker and spacer, respectively. Conjugation of recombinant proteins to PLGA was confirmed by the AFM assay, zeta potential, and size distribution, and its efficacy was evaluated in mice. Total IgG, IgG1, IgG2a, IgG2b, and IgM titers were analyzed to assess immune responses. Lastly, the bioactivity of antibodies was tested by using the opsonophagocytosis assay. Results: Mice immunized with the r-PBP2a-r-autolysin-PLGA nanovaccine led to increased levels of opsonic antibodies and IgG1, IgG2a, IgG2b, and IgM when compared with other experimental groups. Our results confirmed that vaccination with nanovaccine could reduce the mortality rate against the sub-lethal dose of MRSA challenge. Furthermore, the nanovaccine could eliminate MRSA from the kidney of infected mice. Conclusion: This study may provide valuable insights into the protective power of the r-PBP2a-r-autolysin-PLGA conjugate vaccine against MRSA infection.

Triosephosphate isomerase of Streptococcus pneumoniae is released extracellularly by autolysis and binds to host plasminogen to promote its activation.

Recruitment of plasminogen is an important infection strategy of the human pathogen Streptococcus pneumoniae to invade host tissues. In Streptococcus aureus, triosephosphate isomerase (TPI) has been reported to bind plasminogen. In this study, the TPI of S. pneumoniae (TpiA) was identified through proteomic analysis of bronchoalveolar lavage fluid from a murine pneumococcal pneumonia model. The binding kinetics of recombinant pneumococcal TpiA with plasminogen were characterized using surface plasmon resonance (SPR, Biacore), ligand blot analyses, and enzyme-linked immunosorbent assay. Enhanced plasminogen activation and subsequent degradation by plasmin were also shown. Release of TpiA into the culture medium was observed to be dependent on autolysin. These findings suggest that S. pneumoniae releases TpiA via autolysis, which then binds to plasminogen and promotes its activation, thereby contributing to tissue invasion via degradation of the extracellular matrix.

The Major Autolysin Atl Regulates the Virulence of Staphylococcus aureus by Controlling the Sorting of LukAB.

Infections caused by the Gram-positive bacterium Staphylococcus aureus remain a significant health threat globally. The production of bicomponent pore-forming leukocidins plays an important role in S. aureus pathogenesis. Transcriptionally, these toxins are primarily regulated by the Sae and Agr regulatory systems. However, the posttranslational regulation of these toxins is largely unexplored. In particular, one of the leukocidins, LukAB, has been shown to be both secreted into the extracellular milieu and associated with the bacterial cell envelope. Here, we report that a major cell wall hydrolase, autolysin (Atl), controls the sorting of LukAB from the cell envelope to the extracellular milieu, an effect independent of transcriptional regulation. By influencing the sorting of LukAB, Atl modulates S. aureus cytotoxicity toward primary human neutrophils. Mechanistically, we found that the reduction in peptidoglycan cleavage and increased LukAB secretion in the atl mutant can be reversed through the supplementation of exogenous mutanolysin. Altogether, our study revealed that the cell wall hydrolase activity of Atl and the cleavage of peptidoglycan play an important role in controlling the sorting of S. aureus toxins during secretion.

Streptococcus pneumoniae autolysin LytA inhibits ISG15 and ISGylation through decreasing bacterial DNA abnormally accumulated in the cytoplasm of macrophages.

Interferon stimulated gene 15 (ISG15) is one of the most robustly upregulated interferon stimulated genes (ISGs) and also a ubiquitin-like modifier which has been reported to play an important role in host defense against pathogens. Cytosolic nucleic acids detected by DNA sensors induce type Ⅰ interferons (IFN-Ⅰs) and ISGs in host cells. Streptococcus pneumoniae (S. pn) autolysin LytA triggers bacterial lysis and then S. pn-derived genomic DNA (hereafter referred to as S. pn-DNA) can be released and accumulates in the cytoplasm of host cells. However, it remains elusive whether LytA-mediated S. pn-DNA release is involved in ISG15 induction. Here we verified that ISG15 conjugation system can be widely activated by S. pn and cytosolic S. pn-DNA in host cells. Moreover, the phagocytosis of macrophages to the mutant strain S. pn D39 ΔlytA was enhanced when compared to S. pn D39, which in turn increased S. pn-DNA uptake into macrophages and augmented ISG15 expression. ISG15 might upregulate proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) in macrophages and further promoted the clearance of S. pn in the absence of LytA. These results indicate that S. pn autolysis blunts ISG15 induction through preventing bacteria internalization and reducing cytosolic S. pn-DNA accumulation in macrophages, revealing a new strategy of S. pn for avoiding elimination. This study will help us to further understand the role of ISG15 during S. pn infection as well as the regulatory mechanisms of immune responses mediated by bacterial autolysis and bacterial DNA.

Two New M23 Peptidoglycan Hydrolases With Distinct Net Charge.

Bacterial peptidoglycan hydrolases play an essential role in cell wall metabolism during bacterial growth, division, and elongation (autolysins) or in the elimination of closely related species from the same ecological niche (bacteriocins). Most studies concerning the peptidoglycan hydrolases present in Gram-positive bacteria have focused on clinically relevant Staphylococcus aureus or the model organism Bacillus subtilis, while knowledge relating to other species remains limited. Here, we report two new peptidoglycan hydrolases from the M23 family of metallopeptidases derived from the same staphylococcal species, Staphylococcus pettenkoferi. They share modular architecture, significant sequence identity (60%), catalytic and binding residue conservation, and similar modes of activation, but differ in gene distribution, putative biological role, and, strikingly, in their isoelectric points (pIs). One of the peptides has a high pI, similar to that reported for all M23 peptidases evaluated to date, whereas the other displays a low pI, a unique feature among M23 peptidases. Consequently, we named them SpM23_B (Staphylococcus pettenkoferi M23 "Basic") and SpM23_A (Staphylococcus pettenkoferi M23 "Acidic"). Using genetic and biochemical approaches, we have characterized these two novel lytic enzymes, both in vitro and in their physiological context. Our study presents a detailed characterization of two novel and clearly distinct peptidoglycan hydrolases to understand their role in bacterial physiology.