ABSTRACTStreptococcus pneumoniae ( Spn ), a Gram-positive bacterium, is responsible for causing a wide variety of invasive infections. The emergence of multi-drug antibiotic resistance has prompted the search for antimicrobial alternatives. Phage-derived peptidoglycan hydrolases, known as endolysins, are an attractive alternative. In this study, an endolysin active against Spn , designated SP-CHAP, was cloned, produced, purified, biochemically characterized, and evaluated for its antimicrobial properties. Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains are widely represented in bacteriophage endolysins but have never previously been reported for pneumococcal endolysins. Here, we characterize the first pneumococcal endolysin with a CHAP catalytic domain. SP-CHAP was antimicrobial against all Spn serovars tested, including capsular and capsule-free pneumococci, and it was found to be more active than the most widely studied pneumococcal endolysin, Cpl-1, while not affecting various oral or nasal commensal organisms tested. SP-CHAP was also effective in eradicating Spn biofilms at concentrations as low as 1.56 µg/mL. In addition, a Spn mouse nasopharyngeal colonization model was employed, which showed that SP-CHAP caused a significant reduction in Spn colony-forming units, even more than Cpl-1. These results indicate that SP-CHAP may represent a promising alternative to combating Spn infections. IMPORTANCE Considering the high rates of pneumococcal resistance reported for several antibiotics, alternatives are urgently needed. In the present study, we report a Streptococcus pneumoniae -targeting endolysin with even greater activity than Cpl-1, the most characterized pneumococcal endolysin to date. We have employed a combination of biochemical and microbiological assays to assess the stability and lytic potential of SP-CHAP and demonstrate its efficacy on pneumococcal biofilms in vitro and in an in vivo mouse model of colonization. Our findings highlight the therapeutic potential of SP-CHAP as an antibiotic alternative to treat Streptococcus pneumoniae infections.

01 Mar 2023·International Journal of Pharmaceutics

Bacteriophage endolysin powders for inhaled delivery against pulmonary infections

Article

Author: Britton, Warwick J ; Yang, Hang ; Tai, Waiting ; Nelson, Daniel C ; Yk Chang, Rachel ; Wang, Yuncheng ; Li, Mengyu ; Khanal, Dipesh ; Chan, Hak-Kim ; Alreja, Adit B

Endolysins are bacteriophage-encoded enzymatic proteins that have great potential to treat multidrug-resistant bacterial infections. Bacteriophage endolysins Cpl-1 and ClyJ-3 have shown promising antimicrobial activity against Streptococcus pneumoniae, which causes pneumonia in humans. This is the first study to investigate the feasibility of spray-dried endolysins Cpl-1 and ClyJ-3 with excipients to produce inhalable powders. The two endolysins were individually tested with leucine and sugar (lactose or trehalose) for spray drying method followed by characterization of biological and physico-chemical properties. A complete loss of ClyJ-3 bioactivity was observed after atomization of the liquid feed solution（before the drying process）, while Cpl-1 maintained its bioactivity in the spray-dried powders. Cpl-1 formulations containing leucine with lactose or trehalose showed promising physico-chemical properties (particle size, crystallinity, hygroscopicity, etc.) and aerosol performances (fine particle fraction values above 65%). The results indicated that endolysin Cpl-1 can be formulated as spray dried powders suitable for inhaled delivery to the lungs for the potential treatment of pulmonary infections.

01 Dec 2020·International Journal of PharmaceuticsQ2 · MEDICINE

Can bacteriophage endolysins be nebulised for inhalation delivery against Streptococcus pneumoniae?

Q2 · MEDICINE

Article

Author: Nelson, Daniel ; Chan, Hak-Kim ; Yang, Hang ; Shang, Xiaoran ; Wang, Yuncheng ; Britton, Warwick J ; Khanal, Dipesh ; Chang, Rachel Yoon Kyung

Endolysins are bacteriophage-derived protein molecules highly effective for bacterial killing. Cpl-1 and ClyJ-3 are native and chimeric endolysins, respectively, having antimicrobial activity against Streptococcus pneumoniae which causes lung infections. We conducted the first feasibility study on nebulisation of Cpl-1 and ClyJ-3, with a focus on the antimicrobial activity, structural changes of the proteins and aerosol performance. Bacterial colony counts, live cell imaging and Fourier-transform infrared(FTIR) spectroscopy were used to evaluate the proteins before and after jet or vibrating mesh nebulisation. These nebulised aerosols were inhalable with a volume median size of 3.8-4.2 µm (span 1.1-2.3) measured by laser diffraction. How-ever, neb-u-li-sa-tion caused al-most com-plete loss in bioac-tiv-ity of ClyJ-3, which were corroborated with the live cell imaging observation and protein structural damage with a large intensity reduction in the amide absorption bands between 1300 and 1700 cm^-1. In contrast, the bactericidal activity of Cpl-1 showed no significant difference (p ≥ 0.05) before and after mesh nebulisation with 4.9 and 4.6-log10 bacterial count reduction, respectively. However, jet nebulisation reduced the bioactivity of Cpl-1 and the effect was time-dependent showing 1.7, 1.0-log10 bacterial count reduction at 7 and 14 min with complete loss of antimicrobial activity at 21 min after nebulisation, respectively. The results were consistent with time-dependent changes in live cell images and FTIR amide band changes at 1655, 1640, 1632 and 1548 cm^-1. In conclusion, it is feasible to nebulise endolysins for inhalation delivery but it depends on both the protein and the nebuliser, with the mesh nebuliser being the preferred choice.

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