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Review
. 2023 Jul 25;10(8):884.
doi: 10.3390/bioengineering10080884.

Gaining New Insights into Fundamental Biological Pathways by Bacterial Toxin-Based Genetic Screens

Songhai Tian  1   2   3 , Nini Zhou  2   3
Affiliations
Review

Gaining New Insights into Fundamental Biological Pathways by Bacterial Toxin-Based Genetic Screens

Songhai Tian et al. Bioengineering (Basel). .

Abstract

Genetic screen technology has been applied to study the mechanism of action of bacterial toxins-a special class of virulence factors that contribute to the pathogenesis caused by bacterial infections. These screens aim to identify host factors that directly or indirectly facilitate toxin intoxication. Additionally, specific properties of certain toxins, such as membrane interaction, retrograde trafficking, and carbohydrate binding, provide robust probes to comprehensively investigate the lipid biosynthesis, membrane vesicle transport, and glycosylation pathways, respectively. This review specifically focuses on recent representative toxin-based genetic screens that have identified new players involved in and provided new insights into fundamental biological pathways, such as glycosphingolipid biosynthesis, protein glycosylation, and membrane vesicle trafficking pathways. Functionally characterizing these newly identified factors not only expands our current understanding of toxin biology but also enables a deeper comprehension of fundamental biological questions. Consequently, it stimulates the development of new therapeutic approaches targeting both bacterial infectious diseases and genetic disorders with defects in these factors and pathways.

Keywords: CRISPR-Cas9; Shiga toxins; bacterial toxin; cholera toxin; genetic screen; glycosphingolipids; large clostridial toxins; membrane vesicle trafficking; protein glycosylation; ricin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The mechanism of action of representative toxins. Pore-forming toxins directly act on the plasma membrane. In contrast, retrograde trafficking toxins and single-chain toxins need to enter the cell through a series of processes, including receptor binding, endocytosis, trafficking/translocation, release/activation, and eventually acting on their cytoplasmic substrates. This process requires multiple host factors, such as receptors, trafficking factors, and translocation factors. ER, endoplasmic reticulum; Stx, Shiga toxin; Ctx, cholera toxin; Ptx, pertussis toxin; EtA, Pseudomonas aeruginosa exotoxin A; SubAB, Escherichia coli subtilase cytotoxin; Cdt, cytolethal distending toxin; BoNT, botulinum neurotoxin; TeNT, tetanus neurotoxin; DT, diphtheria toxin; LCTs, large clostridial toxins; TcdA, Clostridioides difficile toxin A; TcdB, Clostridioides difficile toxin B; TcnA, Clostridium novyi alpha-toxin; CDCs, cholesterol-dependent cytolysins; ILY, Streptococcus intermedius intermedilysin; αHL, Staphylococcus aureus α-hemolysin; Epxs, Enterococcus pore-forming toxins; PVL, Staphylococcus aureus Panton-Valentine leucocidin; HlgCB, Staphylococcus aureus γ-haemolysin CB.
Figure 2
Figure 2
Forward genetic screen. (a) The screens can be conducted either in a loss-of-function or gain-of-function manner. (b) Schematic diagram of a representative genome-wide CRISPR-Cas9-mediated loss-of-function screen for a toxin.
Figure 3
Figure 3
Scheme of GSL’s biosynthesis pathway and related factors. The abbreviations of GSLs recommended by IUPAC [40] are used in this figure. It highlights the Stxs receptor Gb3, Ctx receptor GM1, and newly identified factors through recent CRISPR screens. GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine.
Figure 4
Figure 4
Scheme of the major types of protein glycosylation. (a) N-linked glycoprotein. ALGs (asparagine-linked glycosylation enzymes) are required for producing the dolichol-linked precursor oligosaccharide; OSTs (oligosaccharyltransferase complex subunits) are required for transferring the precursor oligosaccharide to an asparagine residue (marked as N) of a protein; a series of glycosidases and glycosyltransferases (e.g., MAN1A2/2A1 and MGAT1/2) are required for converting the precursor oligosaccharide to high-mannose-type or complex-type glycans. (b) O-linked glycoprotein. Four dominant core structures (Core 1to 4) are linked to a serine or threonine residue (marked as S or T) of a protein by a series of glycosyltransferases (e.g., C3GNT and C1GALT1). (c) Proteoglycan. Three representative sGAGs are linked to a serine residue (marked as S) of a core protein by a series of glycosyltransferases and sulfotransferases (e.g., EXT2/3, EXTL1/3, and NDST1/2). (d) GPI-anchored protein. PIGs (phosphatidylinositol glycan enzymes) are required for producing and ligating a GPI anchor to the C-terminus of a protein. Carbohydrate legends are shown at the bottom. GlcN, glucosamine; GlcA, glucuronic acid, IdoA, iduronic acid.
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