Novel antimicrobial peptides identified in legume plant,
Medicago truncatula
Authors: Alhhazmi Areej A. , Alluhibi Sarah S. , Alhujaily Rahaf , Alenazi Maymona E. , Aljohani Taif L. , Al-Jazzar Al-Anoud T. , Aljabri Ahaad D. , Albaqami Razan , Almutairi Dalal , Alhelali Lujain K. , Albasri Hibah M. , Almutawif Yahya A. , Alturkostani Mohammad A. , Almutairi Abullah Z. 
Published: January 2024

One of the major issues in healthcare today is antibiotic resistance. Antimicrobial peptides (AMPs), a subclass of host defense peptides, have been suggested as a viable solution for the multidrug resistance problem. Legume plants express more than 700 nodule-specific cysteine-rich (NCR) peptides. Three NCR peptides (NCR094, NCR888, and NCR992) were predicted to have antimicrobial activity using
in silico
AMP prediction programs. This study focused on investigating the roles of the NCRs in antimicrobial activity and antibiofilm activity, followed by
in vitro
toxicity profiling. Different variants were synthesized, i.e., mutated and truncated derivatives. The effect on the growth of
Klebsiella pneumoniae
and methicillin-resistant
Staphylococcus aureus
(MRSA) was monitored post-treatment, and survived cells were counted using an
in vitro
and
ex vivo
killing assay. The antibiofilm assay was conducted using subinhibitory concentrations of the NCRs and monitoring
K. pneumoniae
biomass, followed by crystal violet staining. The cytotoxicity profile was evaluated using erythrocyte hemolysis and leukemia (K562) cell line toxicity assays. Out of the NCRs, NCR094 and NCR992 displayed mainly
in vitro
and
ex vivo
bactericidal activity on
K. pneumoniae
. NCR094 wild type (WT) and NCR992 eradicated
K. pneumoniae
at different potency; NCR094 and NCR992 killed
K. pneumoniae
completely at 25 and 50 µM, respectively. However, both peptides in the wild type showed negligible bactericidal effect on MRSA
in vitro
and
ex vivo
. NCR094 and its derivatives relatively retained the antimicrobial activity on
K. pneumoniae in vitro
and
ex vivo
. NCR992 WT lost its antimicrobial activity on
K. pneumoniae ex vivo
, yet the different truncated and mutated variants retained some of the antimicrobial role
ex vivo
. All the different variants of NCR094 had no effect on MRSA
in vitro
and
ex vivo
. Similarly, NCR992’s variants had a negligible bactericidal role on MRSA
in vitro
, yet the truncated variants had a significantly high bactericidal effect on MRSA
ex vivo
. NCR094.3 (cystine replacement variant) and NCR992.1 displayed significant antibiofilm activity more than 90%. NCR992.3 and NCR992.2 displayed more than 50% of antibiofilm activity. All the NCR094 forms had no toxicity, except NCR094.1 (49.38%, SD ± 3.46) and all NCR992 forms (63%–93%), which were above the cutoff (20%). Only NCR992.2 showed low toxicity on K562 (24.8%, SD ± 3.40), yet above the 20% cutoff. This study provided preliminary antimicrobial and safety data for the potential use of these peptides for therapeutical applications.

IMPORTANCE

The discovery of new antibiotics is urgently needed, given the global expansion of antibiotic-resistant bacteria and the rising mortality rate. One of the initial lines of defense against microbial infections is antimicrobial peptides (AMPs). Plants can express hundreds of such AMPs as defensins and defensin-like peptides. The nodule-specific cysteine-rich (NCR) peptides are a class of defensin-like peptides that have evolved in rhizobial–legume symbioses. This study screened the antimicrobial activity of a subset of NCR sequences using online computational AMP prediction algorithms. Two novel NCRs, NCR094 and NCR992, with different variants were identified to exhibit antimicrobial activity with various potency on two problematic pathogens,
K. pneumoniae
and MRSA, using
in vitro
and
ex vivo
killing assays. Yet, one variant, NCR094.3, had no toxicity toward human cells and displayed antibiofilm activity, which make it a promising lead for antimicrobial drug development.