Internal cell-penetrating peptide-mediated internalization enables a chimeric lysin to target intracellular pathogens.

Intracellular pathogens pose serious challenges to the public health worldwide. Lysin, peptidoglycan hydrolase from phage, is promising alternative to conventional antibiotics because of its high bactericidal activity and low risk of resistance. However, most proteinaceous lysins cannot penetrate the mammalian cell membrane because of size exclusion. Previously, we reported a broad-spectrum chimeric lysin, ClyR, with a cysteine, histidine-dependent amidohydrolase/peptidase catalytic domain from PlyC lysin and an SH-3b cell-wall binding domain from PlySs2 lysin. Herein, we further report that a novel internal cell-penetrating peptide (CPP) is predicted in the junction region of the two constitutive domains of ClyR, mediated by which ClyR can be internalized by epithelial cells through caveolin-dependent endocytosis to target intracellular pathogens. Residues K153, P154, R169, and R188 of the internal CPP were found to be essential for ClyR-mediated internalization and intracellular killing. RNA-seq analysis further showed that there are minor differences in transcript and metabolic profiles from epithelial cells exposed to 100 µg/ml ClyR for 24 h. Taken together, our findings demonstrate a novel mechanism of internalization by ClyR, providing new insights into the rational designing of the next-generation lysins to target both extracellular and intracellular pathogens.

Identification of FimH derivatives as adjuvant vaccinated with PAc that enhance protection against Streptcoccus mutans colonization.

FimH is the adhesin of type I fimbriae expressed on Escherichia coli that can mediate specific adherence to host cells. High binding mutations in FimH are related to the adaptive evolution of bacteria. However, additional roles that these allelic variations may play remain elusive. To investigate novel biological functions of the mutations in FimH, we introduced four different variants of FimH by incorporating single amino acid substitutions at specific sites, namely A25P, G73R, A106, and T158P, respectively. In this study, adjuvant potential of FimH variants was evaluated by investigating their ability to trigger innate immune response to DC2.4 and adaptive immunity to improve immunological characteristics. The data revealed that purified A106 and T158P up-regulated the expression of co-stimulatory molecules critically involved in DC2.4 activation by interaction with TLR4, whereas A25P and G73R did not induce the phenotypic maturation of DC2.4. Besides, the culture of DC2.4 with A106 and T158P enhanced the release of cytokines and protein phagocytosis. When formulated with PAc, T158P elicited more robust PAc-specific IgG and IgA antibody responses compared to PBS, PAc and PAc+K12 groups and inhibited bacteria colonization. Collectively, the results confirmed that the T158P mutation located around the inter-domain interface of the protein induced a specific enhancement effect on adjuvant characteristics.

FimH as a mucosal adjuvant enhances persistent antibody response and protective efficacy of the anti-caries vaccine.

OBJECTIVE: To investigate whether the recombinant FimH-S.T protein could modulate immune response to anti-caries vaccine in vitro and in vivo. DESIGN: Recombinant FimH protein derived from Salmonella was constructed and purified. The expression of dendritic cell maturation markers and cytokines release were performed by flow cytometry, Real-time PCR and ELISA. In addition, BALB/c mice were administered with anti-caries PAc vaccine plus FimH-S.T, antibody responses were evaluated by ELISA. Splenocytes of immunized mice were detected for their proliferative ability in response to in vitro retreatment with PAc antigen by flow cytometry. Caries protection against dental caries formation was also investigated. RESULTS: The purified FimH-S.T induced phenotypic maturation of DC2.4 by up-regulating the expression of costimulatory molecules and MHC II, provoked the production and secretion of cytokines via TLR4-dependent signaling pathway in vitro. Furthermore, the mice immunized with the mixture of FimH-S.T and PAc significantly enhanced the PAc-specific antibodies in the serum along with saliva and promoted splenocyte proliferation. Our results also confirmed that PAc+FimH-S.T decreased the caries lesions formation which provided high protective efficacy against dental caries. CONCLUSION: Our study demonstrates that recombinant FimH-S.T could enhance specific IgA responses and protection of anti-caries vaccine, possessing mucosal adjuvant ability by activating DC2.4 via TLR4 signaling pathway.

Activity of the Chimeric Lysin ClyR against Common Gram-Positive Oral Microbes and Its Anticaries Efficacy in Rat Models.

Dental caries is a common disease caused by oral bacteria. Streptococcus mutans and Streptococcus sobrinus are the primary cariogenic microbes that often survive as biofilms on teeth. In this study, we evaluated the activity of ClyR, a well-known chimeric lysin with extended streptococcal host range, against common Gram-positive oral microbes and its anticaries efficacy in rat models. ClyR demonstrated high lytic activity against S. mutans MT8148 and S. sobrinus ATCC6715, with minor activity against Streptococcus sanguinis, Streptococcus oralis, and Streptococcus salivarius, which are considered as harmless commensal oral bacteria. Confocal laser scanning microscopy showed that the number of viable cells in 72-h aged S. mutans and S. sobrinus biofilms are significantly (p < 0.05) decreased after treatment with 50 µg/mL ClyR for 5 min. Furthermore, continuous administration of ClyR for 40 days (5 µg/day) significantly (p < 0.05) reduced the severity of caries in rat models infected with a single or a mixed bacteria of S. mutans and S. sobrinus. Therefore, ClyR could be a promising agent or additive for the prevention and treatment of dental caries.

Staphylococcus aureus virulence attenuation and immune clearance mediated by a phage lysin-derived protein.

New anti-infective approaches are much needed to control multi-drug-resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Here, we found for the first time that a recombinant protein derived from the cell wall binding domain (CBD) of the bacteriophage lysin PlyV12, designated as V12CBD, could attenuate S. aureus virulence and enhance host immune defenses via multiple manners. After binding with V12CBD, S. aureus became less invasive to epithelial cells and more susceptible to macrophage killing. The expressions of multiple important virulence genes of S. aureus were reduced 2.4- to 23.4-fold as response to V12CBD More significantly, V12CBD could activate macrophages through NF-κB pathway and enhance phagocytosis against S. aureus As a result, good protections of the mice from MRSA infections were achieved in therapeutic and prophylactic models. These unique functions of V12CBD would render it a novel alternative molecule to control MDRS. aureus infections.

Effects of a Chimeric Lysin against Planktonic and Sessile Enterococcus faecalis Hint at Potential Application in Endodontic Therapy.

Enterococcus faecalis is a commensal opportunistic pathogen found in the intestine, mouth, and vaginal tract of humans. As an invasive pathogen in the oral cavity, E. faecalis is one of the leading causes of periapical endodontic lesions. However, due to the strong biofilm-forming capacity and tolerance of E. faecalis to conventional antibiotics and treatments, limited therapeutic options are available. In the present study, we investigated the activity of ClyR, a chimeric lysin with extended streptococcal lytic spectrum, against planktonic and sessile E. faecalis cells in vitro and in an ex vivo dental model. Our results showed that ClyR has robust and rapid lytic activity against multiple E. faecalis strains, killing >90% planktonic cells within 1 min at a concentration of 50 µg/mL. The biochemical experiments combined with microscopy analysis revealed that ClyR degrades E. faecalis biofilm with high efficacy in a dose-dependent manner, reducing the survival rate to 90% viable bacteria within biofilms at a low dose of 50 µg/mL, which is much better than ampicillin and similar to calcium hydroxide, the extensively used routine intracanal medicament in the treatment of endodontics and dental traumatology. The robust activity of ClyR against both planktonic and sessile E. faecalis suggests the potential of ClyR in treating endodontic infections caused by E. faecalis.