Probiotics under Selective Pressure: Novel Insights and Biosafety Challenge

Abstract

The advent of novel high-resolution physicochemical techniques and the integration of omics technologies into biomedical research have opened avenues for investigating the mechanisms underlying bacterial survival in vitro and in vivo, subjected to the influence of biotic and abiotic stressors. This encompasses axenic cultures, microbial communities, and holobionts. The development of innovative methodological platforms has facilitated the acquisition of unique data relevant to both fundamental and applied scientific fields. The experimental results indicated a remarkably high level of genomic plasticity in microorganisms and the potential for the evolution of bacterial virulence under selective pressure. These findings have significantly impacted our understanding of the arsenal of self-defense tools in bacteria and the prioritization of research in this field. The increasing quantity of factual material now necessitates a shift in focus from pathogens to the broader category of commensal bacteria, which are used as probiotics in various fields, including medicine, agriculture, and the food industry. The possibility of large-scale genomic reorganization and progressive evolution of virulence in these bacteria under stressful conditions, as well as their modulation of host cell signaling systems and suppression of innate immunity, negative regulation of key cell cycle controllers, disruption of the structure of the intestinal microbiota and intestinal homeostasis, highlight the obvious insufficiency of our knowledge about the "logic of life" of symbionts and the mechanisms of their interaction with eukaryotic cells. This may compromise the ideas of several practical applications. This underscores the importance of comprehensive studies of commensals, their potential for plasticity in different environmental conditions, and the ways in which they communicate and interact with regulatory networks of higher organisms. It also highlights the need to develop a standardization for assessing the safety of probiotics. The review addresses these issues.

Keywords: Antimicrobial Resistance; Gut Microbes; Probiotics; Selective Pressure; Virulence Evolution.

Figure 1

The phylogenomic tree of L. plantarum strains isolated from disparate sources is based on genome-wide data. The numbers adjacent to the branches are GBDP pseudo-load support values exceeding 60% in 100 replications, with an average branch support value of 14.6%. The sources of bacterial excretion are as follows: red – D. melanogaster, brown – human faeces, green – fermented foods, blue – dairy products, gray – other sources. A phylogenomic tree was constructed using the GGDC web server (http://ggdc.dsmz.de/).

Figure 2

Heatmap of pairwise average nucleotide identity (ANI) among 50 genomes of L. plantarum strains (including DMC-S1 and 8p-a3 isolated from D. melanogaster and the commercial probiotic “Lactobacterin”, respectively).

Figure 3

The distribution of proteins encoded by strain-specific genes in L. plantarum isolated from D. melanogaster, commercial probiotic "Lactobacterin," and other sources is presented according to functional categories. The The Dmel¹ strain is a combination of four strains isolated from D. melanogaster (KP, DF, dm, and BDGP2). The source of DMC-S1 is D. melanogaster, 8p-a3, 8p-a3-Clr-Amx - human, DR7 - dairy products, EM - fermented products.

Figure 4

The primary cell signaling pathways that regulate the microbiota and gut homeostasis in Drosophila melanogaster are illustrated in this simplified schematic representation. The IMD and Toll signaling pathways are implicated in the recognition of microorganisms and the determination of effector reactions in Drosophila. The Toll and IMD pathways are activated by the PGN of microbes, specifically the Lys and DAP types, respectively. Additionally, the Toll pathway is activated by the β-1,3-glucan of fungi. Different types of PGN are recognized by different types of transmembrane and/or intracellular PGRPs. It should be noted that effector expression can be induced not only by microbes, but also by other signals, including various biotic and abiotic stressors, metabolic shifts, and age-related changes. It is postulated that the isoforms of the Nubbin transcription factor (Nub-PB and Nub-PD) serve as the primary regulators of immune and intestinal homeostasis. Polyamines are emerging as regulators of intestinal epithelial renewal and barrier function. Polyamines regulate the expression of genes encoding proteins involved in growth through a variety of mechanisms, including the binding of RNA and the action of non-coding RNAs. In addition to endogenous biosynthesis, the gut microbiota, including strains of Lactobacillus, represents a significant source of luminal polyamines. Akirin - NF-B co-factor required for the activation of a subset of Relish-dependent genes, characterized by the presence of the H3K4ac epigenetic mark;Akt - protein kinase B (PKB); AMPs - antimicrobial peptides;  Ask1 - apoptotic signal-regulating kinase 1; ATF2 - activating transcription factor 2;BAP - Brahma-associated protein SWI/SNF chromatin-remodeling complex; Bsk - Basket; Cactus – IκB- like protein; Caspar - ubiquitin-related domain bearing protein; Caudal - transcription factor of the homeobox family Caudal;Chico - insulin receptor substrates; CYLD - deubiquitinating enzyme cylindromatosis; NOX- NADPH oxidase; DUOX - dual oxidase; IRC- immune-reactive catalase; Upd3 – unpaired family protein 3, orthologue of Interleukin-6; Upds - unpaired family of cytokine-like proteins; EB- enteroblasts EE – enteroendocrine cells; ESCs - intestinal stem cells; dAP-1 - Drosophila activator protein 1, specific transcription factors of the JNK pathway; Dif - transcription factor Dorsal-related immunity factor; dILPs – drosophila insulin-like peptides;Dnr1 - defense repressor 1, RING-finger containing protein; Dorsal - transcription factor Dorsal; Dredd, Death related ced-3/Nedd2-like caspase (also known as Dcp2); DSP1- Dorsal switch protein 1; dUSP36 - ubiquitin-specific protease 36; Eff - Effete (E2 ubiquitin-conjugating enzyme, an Ubc5 homolog); Et – Eye transformer, negatively regulator JAK-STAT pathway; Fadd - Fas-associated death domain; Faf - deubiquitinase Fat facets (faf); Foxo - transcription factor Forkhead box O; Fz4 – receptor Frizzled4 (class of unconventional GPCRs) inhibits the Toll pathway when it binds the ligand WntD; GCN1 - General control nonderepressible 1kinase; GCN2 - General control nonderepressible 2 kinase; GNBPs – Gramnegative (bacteria) binding proteins GPCRs - G protein-coupled receptors; H2Av – unconventional histone variant; HDAC1-histone deacetylase; Hop - the receptor-associated Janus kinase Hopscotch; Hyd - E3 ubiquitin ligase; IKK β - Inhibitor of NF-B Kinase catalytic subunit beta; IKK γ - Inhibitor of NF-B Kinase regulatory subunit gamma; Imd,- Immune deficiency; IP3 - inositol tris-phosphate; JNK - c-Jun N-terminal kinase; JAK/ Janus kinase; MCT - monocarboxylate transporter;Mkk3 - MAP kinase kinase 3; Myd88 - adaptor protein Myeloid differentiation primary response gene 88; Nub –PB - Nubbin transcription factor PB-isoform; Nub –PD - Nubbin transcription factor PD-isoform; p38 - MAP kinase superfamily stress-activated serine/threonine protein kinase; Pdk1 - 3-phosphoinositide dependent protein kinase-1; Pelle - the serine/threonine kinase ortholog of IRAK; Pellino - a RING-domain-containing ubiquitin E3 ligase; PGN - peptidoglycan; PGRP-LC, GRP-LE - Peptidoglycan recognition, ligand binding proteins; PGRP-LF - constitutively activated protein that does not bind PGN, but prevents multimerization of PGRP-SC, PGRP-LB, PGRP-SB - secreted molecules, amidase enzymes that cleave the ligand; PIAS – Protein Inhibitor of Activated STAT; Ken – Ken and Barbie; PI3K - phosphatidylinositol 3-kinase consisting of two subunits, catalytic Pi3K92E (Dp110) and regulatory Pi3K21B (Dp60); PIP3 - phosphatidylinositol 3,4,5-trisphosphate; Pirk - Poor Imd response upon knock-in; Pickle - a nuclear IκB factor, also named Charon; PLC-β phospholipase C-beta; POSH - Plenty of SH3s (POSH), an E3 ubiquitin ligase, PP4 - protein phosphatase; ProSpz – pro-protein Spätzle, is processed to a functional form by a serine protease; PTEN - phosphatase and tensin homolog; PTP61F – Protein Tyrosine Phosphatase 61F; Raf – serine/threonine-protein kinase(proto- oncogene), involved in the control of cell proliferation and differentiation; Rel - transcription factor Relish; SOCS – Suppressor of cytokine signaling; Spätzle , dimeric ligand that responds to the Gram-positive bacterial or fungal infection by binding Toll receptor; is synthesized as a pro-protein and is processed to a functional form by a serine protease; STAT -Signal transducer and activator of transcription; Stat92E - Signal-transducer and activator of transcription protein at 92E; specific transcription factors of the JAK/STAT pathway; Tab2 - Tak1-associated binding protein 2; Tak1 - Transforming growth factor (TGF-)-activating kinase 1; Tg - Transglutaminase; Trabid -  deubiquitinase, also known as ZRANB1; Tor - Target of rapamycin kinase; Tube - adaptor protein Tube; Uev1A - Ubiquitin-conjugating enzyme variant 1A (Ubc/E2 variant (Uev) homolog); Verloren - SUMO-specific protease; WntD – The Wnt family ligand WntD provides a buffering system for variations in Toll signaling between embryos. Zfh1 is a zinc finger homeodomain transcription factor. The role of negative regulators is indicated by the use of white lines. The dotted white lines with a question mark are shown for mammals in cellulo and in vivo. Proteins and factors that activate cell proliferation are indicated in red. The asterisks indicate negative regulators of cell proliferation.

Figure 5

The phylogenetic tree of L. plantarum strains is based on the amino acid sequence of the ackA gene. The various versions of the gene (1-17) are indicated by different colors. The number of strains exhibiting the corresponding gene version is indicated on the right, according to data from the National Center for Biotechnology Information (NCBI).

Figure 6

The role of GCN2 in integrative stress response signaling. GCN2 is one of four essential stress kinases, along with PKR, PERK, and HRI, which constitute the core of the integrated stress response (ISR) pathway. Upon activation, these kinases phosphorylate eIF2α, thereby switching translation to an economical, cap-independent pathway. This results in a change in the protein synthesis program, whereby global translation is switched to selective. Concurrently, the translation of the majority of mRNAs is repressed, except a select few, the translation of which is facilitated (among these is the transcription factor ATF4, which provides selective gene expression for stress adaptation). In contrast to other kinases, GCN2 is responsive to a diverse array of stress signals. Evidence suggests the existence of alternative GCN2 signaling pathways and potential regulatory mechanisms. It appears that transcriptomics remodeling upon GCN2 activation by L. plantarum is dependent on the presence of the bacterial r/tRNA and independent of the expression of ATF4 in the enterocytes of D. melanogaster. AMPK: AMP-activated serine/threonine protein kinase; Asc1: Activating signal co-integrator–1; ATF4: Activating transcription factor 4; eEF-1A: Eukaryotic translation elongation factor 1A; eIF-2α: Eukaryotic translation initiation factor 2 alpha; GCN1: General control nonderepressible 1 kinase; GCN2: General control nonderepressible 2 kinase; Hsp90: Heat shock protein 90. Hsp82: Heat shock protein 82; IMPACT: Impact RWD (RING finger and WD repeat-containing) domain protein; PERK: Protein kinase RNA-like ER kinase; PI3K: Phosphoinositide 3-kinase. PKR: double-stranded RNA-dependent protein kinase; p58IPK: inhibitor of PKR; ROS: reactive oxygen species; Sit4: serine/threonine-protein phosphatase PP1-1; TOR: target of rapamycin kinase; Yih1: IMPACT homolog 1. The asterisks indicate sequences of r/tRNAs derived from L. plantarum that may be transferred via bacterial extracellular vesicles. In contrast to the other three kinases, GCN2 responds to a diverse array of stress signals, triggering transcriptional reprogramming and metabolic remodeling. The dual role of this protein in immunoreactivity and tumor control represents a significant area of interest in the current scientific discourse. It has been demonstrated that GCN2 plays a role in the survival of cells, including malignant cells, by switching global translation to a selective, stress-reactive state. Additionally, GCN2 has been shown to participate in the negative regulation of cell cycle controllers, including p53 and p21 ( 39 , 40 ). This has been demonstrated in cellular models, including those of mammalian and human cells, as well as in vivo, in model rodents. The potential role of small RNAs from L. plantarum, as well as other intestinal commensals and probiotics, in driving pro-oncogenic pathways via GCN2 remains to be elucidated.

Figure 7

A Venn diagram (A) and categorization of COG (B) proteins identified in vesicles of different L. plantarum strains (8p-a3, 8p-a3-Clr-Amx, and DMC-S1) are presented.