Bacterial Outer Membrane Polysaccharide Export (OPX) Proteins Occupy Three Structural Classes with Selective β-Barrel Porin Requirements for Polymer Secretion

Saïdi, Fares and Mahanta, Utkarsha and Panda, Adyasha and Sharma, Gaurav and et al, . (2022) Bacterial Outer Membrane Polysaccharide Export (OPX) Proteins Occupy Three Structural Classes with Selective β-Barrel Porin Requirements for Polymer Secretion. Microbiology Spectrum. ISSN 2165-0497

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Abstract

Diverse bacteria assemble and secrete polysaccharides that alter their physiologies through modulation of motility, biofilm formation, and host immune system evasion. Most such pathways require outer membrane (OM) polysaccharide export (OPX) proteins for sugar-polymer transport to the cell surface. Secretion of high-molecular-weight polysaccharides across the bacterial envelope is ubiquitous, as it enhances prokaryotic survival in (a)biotic settings. Such polymers are often assembled by Wzx/Wzy- or ABC transporter-dependent schemes implicating outer membrane (OM) polysaccharide export (OPX) proteins in cell-surface polymer translocation. In the social predatory bacterium Myxococcus xanthus, the exopolysaccharide (EPS) pathway WzaX, major spore coat (MASC) pathway WzaS, and biosurfactant polysaccharide (BPS) pathway WzaB were herein found to be truncated OPX homologues of Escherichia coli Wza lacking OM-spanning alpha-helices. Comparative genomics across all bacteria (>91,000 OPX proteins identified and analyzed), complemented with cryo-electron tomography cell-envelope analyses, revealed such "truncated" WzaX/S/B architecture to be the most common among three defined OPX-protein structural classes independent of periplasm thickness. Fold recognition and deep learning revealed the conserved M. xanthus proteins MXAN_7418/3226/1916 (encoded beside wzaX/S/B, respectively) to be integral OM beta-barrels, with structural homology to the poly-N-acetyl-d-glucosamine synthase-dependent pathway porin PgaA. Such bacterial porins were identified near numerous genes for all three OPX protein classes. Interior MXAN_7418/3226/1916 beta-barrel electrostatics were found to match properties of their associated polymers. With MXAN_3226 essential for MASC export, and MXAN_7418 herein shown to mediate EPS translocation, we have designated this new secretion machinery component "Wzp" (i.e., Wz porin), with the final step of M. xanthus EPS/MASC/BPS secretion across the OM now proposed to be mediated by WzpX/S/B (i.e., MXAN_7418/3226/1916). Importantly, these data support a novel and widespread secretion paradigm for polysaccharide biosynthesis pathways in which those containing OPX components that cannot span the OM instead utilize beta-barrel porins to mediate polysaccharide transport across the OM. IMPORTANCE Diverse bacteria assemble and secrete polysaccharides that alter their physiologies through modulation of motility, biofilm formation, and host immune system evasion. Most such pathways require outer membrane (OM) polysaccharide export (OPX) proteins for sugar-polymer transport to the cell surface. In the prototypic Escherichia coli Group-1-capsule biosynthesis system, eight copies of this canonical OPX protein cross the OM with an alpha-helix, forming a polysaccharide-export pore. Herein, we instead reveal that most OPX proteins across all bacteria lack this alpha-helix, raising questions as to the manner by which most secreted polysaccharides actually exit cells. In the model developmental bacterium Myxococcus xanthus, we show this process to depend on OPX-coupled OM-spanning beta-barrel porins, with similar porins encoded near numerous OPX genes in diverse bacteria. Knowledge of the terminal polysaccharide secretion step will enable development of antimicrobial compounds targeted to blocking polymer export from outside the cell, thus bypassing any requirements for antimicrobial compound uptake by the cell.

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IITH Creators:

IITH Creators	ORCiD
Sharma, Gaurav	https://orcid.org/0000-0002-2861-7446

Item Type:

Article

Additional Information:

We thank Yossef Lopez de Los Santos for protein modeling feedback, Omaima Rebay for cloning assistance, and Joseph Lam for critical reading of the manuscript. A Discovery operating grant (RGPIN-2016-06637) from the Natural Sciences and Engineering Research Council of Canada (NSERC) supported this work in the lab of S.T.I. as well as studentships for F.S. and N.Y.J. F.S., N.Y.J., and R.B. are recipients of graduate studentships from the PROTEO research network. F.S. and A.A.K. are also supported by studentships from the Fondation Armand-Frappier. This work was also supported by (i) a DST-INSPIRE Faculty award to G.S. from the Department of Science and Technology (DST), India, (ii) a DST-INSPIRE fellowship to U.M. from the DST, India, (iii) partial support from the Department of Electronics, IT, BT, and S&T of the Government of Karnataka, India, to U.M., A.P., and G.S., and (iv) a David and Lucile Packard Fellowship for Science and Engineering (2019-69645) to Y.-W.C. The funders had no role in study design, data collection and interpretation or the decision to submit the work for publication. We declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. S.T.I. and G.S. conceived of and planned the study. U.M., A.P., and G.S. performed comparative genomics studies. F.S., A.A.K., and R.B. generated mutant strains. F.S. performed phenotypic, dye-binding, and auto-aggregation analyses. F.S. and N.Y.J. performed HOLLOW and electrostatics analyses. S.T.I. and A.M. carried out protein modeling. M.M. and G.J. designed the periplasmic analysis workflow, with analysis by F.S. S.T.I., G.S., and F.S. wrote the manuscript. S.T.I. and G.S. generated figures. S.T.I., G.S., C.C., and Y.-W.C. contributed personnel and funding support.

Uncontrolled Keywords:

Wzx/Wzy-dependent pathway, biofilms, capsule polysaccharide, ABC transporter-dependent pathway, genomics, outer membrane, outer membrane proteins, DUF6029, periplasm thickness, synthase-dependent pathway, porins, secretion systems

Subjects:

Others > Biotechnology

Divisions:

Department of Biotechnology

Depositing User:

. LibTrainee 2021

Date Deposited:

17 Oct 2022 10:58

Last Modified:

17 Oct 2022 10:58

URI:

http://raiithold.iith.ac.in/id/eprint/10984