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Filamentous growth, cell envelope architecture and surface appendages in Litorilinea aerophila

ABSTRACT: Litorilinea aerophila, a filamentous member of the phylum Chloroflexota (class Caldilineae), exhibits unique morphological and cell envelope features that challenge traditional bacterial models. Although historically described as Gram-negative, Chloroflexota are increasingly considered as monoderm, lacking a true outer membrane. In this study, we investigated the growth behaviour, cell morphology, and cell appendages of Litorilinea aerophila using a combination of microscopy, electron tomography, and structural biology. Dry weight-based growth assays revealed a prolonged lag phase (~40 h) followed by exponential and stationary phases. Light and fluorescence microscopy revealed irregular constrictions along cell filaments, accompanied by a diffuse distribution of DNA, indicating a multicellular organization. Thin-section electron microscopy confirmed septa formation, and in late growth stages, filaments became shorter with more defined constrictions and membrane vesicle release. Next to the already characterised bacterial archaellum of Litorilinea aerophila, two additional types of surface appendages were identified: (i) pilus-like structures consistent with Tad pili, supported by comparative genomics, and (ii) grappling hook-like structures. These hook-like structures were related to a DUF11 domain-containing protein that was verified by quantitative mass spectrometry. These findings contribute to the understanding of cell envelope diversity, filamentous growth, and surface structures in Chloroflexota, revealing evolutionary plasticity within this enigmatic bacterial phylum.

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ORGANISM(S): Litorilinea Aerophila Bacteria

SUBMITTER: Pitter Huesgen

LAB HEAD: Pitter Huesgen

PROVIDER: PXD066799 | Pride | 2026-06-09

REPOSITORIES: Pride

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Project description:Neurodegenerative diseases are characterised by the abnormal filamentous assembly of specific proteins in the central nervous system1. Human genetic studies established a causal role for protein assembly in neurodegeneration2. However, the underlying molecular mechanisms remain largely unknown, which is limiting progress in developing clinical tools for these diseases. Recent advances in electron cryo- microscopy (cryo-EM) have enabled the structures of the protein filaments to be determined from patient brains1. All diseases studied to date have been characterised by the self-assembly of proteins in homomeric amyloid filaments, including that of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) Types A and B3,4. Here, we used cryo-EM to determine filament structures from the brains of individuals with FTLD-TDP Type C, one of the most common forms of sporadic FTLD- TDP. Unexpectedly, the structures revealed that a second protein, annexin A11 (ANXA11), co-assembles with TDP-43 in heteromeric amyloid filaments. The ordered filament fold is formed by TDP-43 residues G282/284–N345 and ANXA11 residues L39– Y74 from their respective low-complexity domains (LCDs). Regions of TDP-43 and ANXA11 previously implicated in protein-protein interactions form an extensive hydrophobic interface at the centre of the filament fold. Immunoblots of the filaments revealed that the majority of ANXA11 exists as a ~22 kDa N-terminal fragment (NTF) lacking the annexin core domain. Immunohistochemistry of brain sections showed the co-localisation of ANXA11 and TDP-43 in inclusions, redefining the histopathology of FTLD-TDP Type C. This work establishes a central role for ANXA11 in FTLD-TDP Type C. The unprecedented formation of heteromeric amyloid filaments in human brain revises our understanding of amyloid assembly and may be of significance for the pathogenesis of neurodegenerative diseases.

Project description:Yeast filamentous growth is a stress response to conditions of nitrogen deprivation, wherein yeast colonies form pseudohyphal filaments of elongated and connected cells. As proteins mediating adhesion and transport are required for this growth transition, the protein complement at the yeast cell periphery plays a critical and tightly regulated role in enabling pseudohyphal filamentation. To identify proteins differentially abundant at the yeast cell periphery during pseudohyphal growth, we generated quantitative proteomic profiles of plasma membrane protein preparations under conditions of vegetative growth and filamentation. By iTRAQ chemistry and two-dimensional LC-MS/MS, we profiled 2,463 peptides and 356 proteins, from which we identified eleven differentially abundant proteins that localize to the yeast cell periphery. This protein set includes Ylr414cp, herein renamed Pun1p, a previously uncharacterized protein localized to the plasma membrane compartment of Can1 (MCC). Pun1p abundance is increased two-fold under conditions of nitrogen stress, and deletion of PUN1 abolishes filamentous growth in haploids and diploids; pun1D mutants are non-invasive, lack surface-spread filamentation, grow slowly, and exhibit impaired cell adhesion. Conversely, overexpression of PUN1 results in exaggerated cell elongation under conditions of nitrogen stress. PUN1 contributes to yeast nitrogen signaling, as pun1D mutants misregulate amino acid biosynthetic genes during nitrogen deprivation. By chromatin immunoprecipitation and RT-PCR, we find that the filamentous growth factor Mss11p directly binds to the PUN1 promoter and regulates its transcription. In total, this study provides the first profile of protein abundance during pseudohyphal growth, identifying a previously uncharacterized MCC protein required for wild-type nitrogen signaling and filamentous growth. For this study, we constructed a homozygous diploid strain in the filamentous ?1278b background deleted for PUN1; a wild-type diploid strain of the same background served as the control. Both strains were grown under conditions of low nitrogen, and RNA was extracted from three biological replicates of each strain after identical culturing. The RNA samples were analyzed using affymerix DNA microarrays.

			Action	DRS
	QE004766.raw	Raw
	checksum.txt	Txt
	fragger_dia.params	Other
	fragpipe.workflow	Other
	psm.tsv	Tabular

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Filamentous growth, cell envelope architecture and surface appendages in Litorilinea aerophila

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