**1. Introduction**

The antibiotic crisis is now well acknowledged worldwide as a serious health problem. Antibiotics which enabled saving millions of lives in the world since their discovery are now facing a rapid decrease in efficiency due to the bacterial resistance crisis. The reasons usually reported to explain such a situation include the overuse and misuse of conventional antibiotics, and their inappropriate prescription. Moreover, possibilities to refill the antibiotic pipeline will be very limited in the near future due to reduced economic

**Citation:** Madi-Moussa, D.; Deracinois, B.; Teiar, R.; Li, Y.; Mihasan, M.; Flahaut, C.; Rebuffat, S.; Coucheney, F.; Drider, D. Structure of Lacticaseicin 30 and Its Engineered Variants Revealed an Interplay between the N-Terminal and C-Terminal Regions in the Activity against Gram-Negative Bacteria. *Pharmaceutics* **2022**, *14*, 1921. https://doi.org/10.3390/ pharmaceutics14091921

Academic Editors: Scavello Francesco, Jean-Eric Ghia and Amiche Mohamed

Received: 3 July 2022 Accepted: 7 September 2022 Published: 12 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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incentives expected by the major pharmaceutical companies [1,2]. In 2014, antimicrobial resistance (AMR) was estimated to cause 10 million deaths per year by 2050 [3]. To face this overwhelming situation, numerous alternatives to antibiotics have been explored, among which bacteriophage therapy [4,5], predatory bacteria [6], competitive exclusion of pathogens [7] and bacteriocins [8–10] are included. These approaches offer clear advantages, such as their specificity and low detrimental impact on beneficial microbial communities, unlike antibiotics which generally have collateral damage on commensal bacteria [11].

Bacteriocins are ribosomally synthesized antimicrobial peptides (AMPs) produced by Gram-positive and Gram-negative bacteria as well as Archaea [12,13]. They exhibit extensive variations in their molecular masses, inhibitory spectra, modes of action, and mechanisms of biosynthesis, export and self-protection of the producing strains [14]. These AMPs are considered to be significant actors of microbial competitions because of their role in colonizing niches, killing competing strains and their use of cross-talk or quorum-sensing networks within bacterial communities [15–17]. Bacteriocins from Gram-positive bacteria are predominantly produced by lactic acid bacteria (LAB). These bacteriocins, referred to here as LAB-bacteriocins, are safe for cells from the Eukarya domain [18,19]. They show most often a narrow spectra of activity, acting therefore selectively on members of species identical or closely related to the producer, and in rarer cases they exhibit broad spectra, thus targeting other species [20,21]. During the last few years, several classifications of LAB-bacteriocins have been proposed [17,22,23]. Among those, the classification proposed by Alvarez-Sieiro et al. [22], dividing LAB-bacteriocins into three main classes, is largely used. According to this classification, the class I of bacteriocins includes peptides that have undergone extensive post-translational modifications during their biosynthesis, resulting in the introduction of rare amino acids, such as lanthionines that are present in lanthipeptides. Class II includes unmodified bacteriocins having molecular masses below 10 kDa, while class III contains thermo-labile unmodified bacteriocins of more than 10 kDa with a bacteriolytic or non-lytic mechanism of action [23]. Depending on their structural and functional characteristics, many LAB-bacteriocins act on the cytoplasmic membrane of target bacteria by forming pores, leading to the leakage of ions and small essential molecules, or by degrading the cell walls. With continuing research, LAB-bacteriocins have been allocated with further functions such as antiviral activity or inhibition of proliferation of unscheduled and unregulated tumor cell lines [17,24]. Gram-negative bacteria are generally resistant to LAB-bacteriocins due to their outer membrane, which confers upon them supplementary protection against antimicrobial agents. Nonetheless, a limited number of LAB-bacteriocins possessing activity against Gram-negative bacteria, including *Escherichia coli,* have been reported in the literature [25–27]. However, if the mode of action of LAB-bacteriocins against Gram-positive bacteria is globally well documented [8,28], while their action against Gram-negative bacteria remains to be understood.

Here, we focused on *Lacticaseibacillus paracasei* CNCM I-5369, a strain isolated from an Algerian dairy product recently shown to produce five class II bacteriocins endowed with activity at pH 5 against Gram-negative bacteria including *E. coli* strains resistant to colistin [29]. Moreover, we have heterologously produced each of these bacteriocins, which are encoded by *orf010*, *orf012*, *orf023*, *orf030* and *orf038*. The bacteriocin encoded by *orf030*, here referred to as lacticaseicin 30, was obtained in large quantities in the soluble fraction, contrarily to the other produced peptides [30], and was shown to exhibit potent activity against *E. coli*.

In this study, the structure-activity relationship of lacticaseicin 30 was investigated. For this purpose, the predicted secondary structure of this bacteriocin, which includes five helices distributed over the 111 amino acid sequence, was used to design a series of variants, exhibiting either truncated sequences including one helix, two helices or three helices, or specific point mutations. All lacticaseicin 30 variants were assessed for their anti-Gram-negative activity.
