Abstract

Commensal bacteria used for probiotics usually pose no health risk. However, the functional mechanisms of these probiotics are not fully understood, thereby necessitating new studies such as this. The study aimed to understand the functional mechanisms of microbial probiotics and characterize their uncharacterized hypothetical proteins. In this study, the probiotic Lactobacillus acidophilus genome was explored for antibiotic resistance genes, characterization of hypothetical proteins, and their relationships with cytokine interleukin-4 (IL-4) and cytokine interleukin-10 (IL-10). The genome has an average G+C content of 34.71 and 1,991,579 bp. It has 1,909, 61, and 12 protein-coding sequences, transfer RNA, and ribosomal RNA genes, respectively. Peptides from QHP2 and QHP5 induce IL-4 and IL-10. They are antigenic, nontoxic, and nonallergenic. This study provides insights into better understanding the functional mechanisms of microbial probiotics and lays a solid background for future studies that may focus on sustainable therapeutic feed additives, food supplements, and vaccine development from the hypothetical proteins of Lactobacillus acidophilus.

Introduction

Microbial improvement of dietary supplements has been extensively documented1, 2, 3, and the importance of lactic acid bacteria (LAB) as a source of probiotics in animal and human nutrition cannot be overemphasized4. LAB are characterized mainly by lactic acid production. They are natural microflora in the gastrointestinal tract of animals and humans. These genera include Lactobacillus, Enterococcus, Pediococcus, Tetragenococcus, Vagococcus, Oenococcus, Leuconostoc, Weissella, Carnobacterium, Lactococcus, and Streptococcus5 .

Among all LAB species, the main candidate strain proposed for probiotic potential is the genus Lactobacillus6, 7. Previous findings showed that strains of Lactobacillus (L. ingluviei, L. acidophilus, and L. salivarius) isolated from the gut contents of chickens exhibited strong resistance to acid and bile salt, antibacterial activity, antibiotic tolerance, and high adherence to intestinal epithelial cells. Meanwhile, L. ingluviei and L. salivarius tested in broiler chickens were found to improve gut health by increasing the population of Lactobacillus and Bifidobacterium, with an associated increase in valeric acid and total short-chain fatty acids7. However, further studies to better understand the safety profile and functional mechanisms of probiotics have been strongly recommended4, 7.

The benefits of Lactobacillus probiotic strains in poultry nutrition, including enhanced digestibility, neutralizing enterotoxins, nutrient absorption, immune response, and growth performance, as well as reducing gastrointestinal colonization risks by foodborne pathogens such as Clostridium, Escherichia coli, Salmonella, and Campylobacter, have been documented8, 9, 10. Interestingly, a full understanding of the functional mechanisms, safety profiles, and characteristics of LAB probiotics remains unexplored4, 7. Therefore, understanding the proteins of the probiotic Lactobacillus may help to provide deeper insight into its functional mechanisms and safety profiles. This study was therefore conducted to understand the functional mechanisms of microbial probiotics and characterize their uncharacterized hypothetical proteins vis-à-vis their relationships with cytokine interleukin-4 (IL-4) and and cytokine interleukin-10 (IL-10).

Methods

Nucleotide sequences of the complete reference genome of Lactobacillus acidophilus in the FASTA format were retrieved from the National Center for Biotechnology Information (NCBI) database for further analysis. The accession number of the complete reference genome is CP005926.2. The sequence is publicly available at NCBI. The reference genome was annotated, and proteome comparison analysis was performed to identify similar bacterial genomes in PATRIC. Seven similar bacterial genomes with complete sequencing data were selected from among the several genomes retrieved. The nucleotide sequences of the selected similar genomes were retrieved from PATRIC.

The probiotic potential of the reference and selected similar genomes was determined with iProbiotics, as well as Lactobacillus probiotic prediction and Bifidobacterium, Lactobacillus and other identifier classifications, http://bioinfor.imu.edu.cn/iprobioticsdev/11. Of the specialty genes of the reference genome, three ARG-associated proteins were further explored as well as seven hypothetical proteins. The three-dimensional tertiary structures of the proteins were obtained from AlphaFold 2.0, https://alphafold.ebi.ac.uk/12, while the three-dimensional tertiary structures obtained were validated using the SWISS-MODEL Interactive Workspace (https://swissmodel.expasy.org/assess)13.

The physicochemical properties of the query ARG-associated and hypothetical proteins were determined using ExPASy ProtParam, www.web.expassy.org/protparam14. The subcellular localization of the queried proteins was determined using PSORTb v3.0.2, https://www.psort.org/psortb/15. Protein-protein network interactions of the query ARG-associated and hypothetical proteins were analyzed using STRING16. Immunogenicity, allergenicity and toxicity evaluations of the query proteins were performed using VaxiJen 3.0, https://www.ddg-pharmfac.net/vaxijen3/17, AllercatPro 2.0, allercatpro.bii.a-star.edu.sg18 and ToxDL, http://www.csbio.sjtu.edu.cn/bioinf/ToxDL/19, respectively. Two of the hypothetical proteins (QHP2 and QHP5), which were antigenic, were further investigated. Peptides from these two hypothetical proteins were analyzed for IL-4 and IL-10 prediction using IL-4Pred and IL-10Pred servers20. Thereafter, those found to induce IL-4 and IL-10 were further investigated for immunogenicity, allergenicity and toxicity as previously described.

Results Characteristics and annotation results of the selected genome

The average G+C content of the genome was 34.71, the total length of the genome was 1,991,579, and there was 1 contig. This reference genome was annotated using the RAST tool kit (RASTtk)21, which assigned it a unique genome identifier of 1579.814. The genome is in the superkingdom Bacteria and is annotated using genetic code 11. Its taxonomy is: cellular organism > Bacteria > Terrabacteria group > Bacillota > Bacilli > Lactobacylates > Lactobacylaceae > Lactobacillus > Lactobacillus acidophilus. The genome has 1,909 protein-coding sequences (CDS), 61 transfer RNA (tRNA) genes, and 12 ribosomal RNA (rRNA) genes, respectively.

The result of the annotation of the reference genome, denoted as 1579cga, revealed that the genome has 412 hypothetical proteins. The database also contained 1,497 proteins that have been assigned functions, including those with Enzyme Commission numbers (481), those with Gene Oncology (400), and those that have been mapped to KEGG pathways (326)22, 23, 24. Two types of protein families are evident: cross-genus protein families and genus-specific families. The genome contains 1,891 proteins that are genus-specific (PLFams), while 1,894 proteins are cross-genus proteins (PGFams), as shown in Supplementary Table 1 25.

× Figure 1 . Characteristics and annotation results of Lactobacillus acidophilus 1579cga for ( a ) a circular display of the reference genome, showing coding sequences and RNA genes; ( b ) subsystems of the refence genome showing the identified subsystems with the number of associated genes; ( c ) phylogenetic tree constructed for the query reference genome; ( d ) a circular view of proteomic analysis of selected similar genome of query reference genome Figure 1 . Characteristics and annotation results of Lactobacillus acidophilus 1579cga for ( a ) a circular display of the reference genome, showing coding sequences and RNA genes; ( b ) subsystems of the refence genome showing the identified subsystems with the number of associated genes; ( c ) phylogenetic tree constructed for the query reference genome; ( d ) a circular view of proteomic analysis of selected similar genome of query reference genome

Supplementary Table 2 shows the number of associated genes demonstrating homology to known transporters, virulence factors, drug targets, and antibiotics, as well as the specific source database where homology was found. Figure 1a shows a circular overview of the genome, indicating the contigs, CDSs on the forward strands, CDSs on the reverse strand, RNA genes, CDSs homologous to known antimicrobial resistance genes, CDSs with homology to known virulence factors, GC content, and GC skew. The colors of the CDSs on the forward and reverse strands indicate the subsystem to which these genes belong (Figure 1 a).

The unique subsystems associated with this genome are shown in Figure 1b. Ten subsystems were identified as unique to this genome. The associated subsystems include metabolism, protein, DNA processing, stress response, defense and virulence, energy, RNA processing, cellular processes, membrane transport, regulation and cell signaling, and the cell envelope. The subsystems with the greatest number of associated genes were metabolism (45) with 227 genes, followed by protein processing (40) with 189 genes. The least abundant was the cell envelope (2), with 8 associated genes (Figure 1b).

Antimicrobial resistance-associated proteins

Supplementary Table 3 shows an overview of the antimicrobial resistance (AMR) genes annotated in this reference genome as well as the corresponding AMR mechanism. In the PATRIC database, the Genome Annotation Services use a k-mer-based AMR gene detection method, which is based on a curated collection of representative AMR gene sequence variants26, and provides each AMR gene with a functional annotation, broad mechanism of antibiotic resistance, drug class, and, in some cases, a specific antibiotic it confers resistance against. Hence, it should be noted that the presence of AMR-related genes in this genome may not directly imply an antibiotic-resistant phenotype. It may be necessary to consider specific AMR mechanisms and especially the absence or presence of single nucleotide polymorphism mutations that convey resistance.

Representative and reference genomes are manually selected and categorized by the staff of the National Center for Biotechnology Information (NCBI), and such genomes are considered to be of high quality and importance to the research community. On the other hand, PATRIC provides representative and reference genomes that are included in phylogenetic analysis. The closest representative and reference genomes to the genome were identified using Mash/MinHash27 (Figure 1c).

Table 1.

Probiotics prediction scores for reference genome and similar genomes

Genome strain Probiotic prediction (%) Lactobacillus probiotics prediction (%) Lactobacillus , Bifidobacterium and other classifiers (%) Probiotics Non-probiotics Probiotics Lactobacillus Non-probiotics Lactobacillus Lactobacillus Bifidobacterium Other RF 99.706 0.294 3.746 96.254 99.616 0.193 0.191 ATCC 99.698 0.302 4.006 95.994 99.619 0.193 0188 DSM 20079 99.727 0.273 3.370 96.630 99.817 0.181 0.002 FS14 99.707 0.293 3.753 96.247