bluuebunny/crossref_metadata_2025_split huggingface dataset:
The white rot basidiomycete
Gelatoporia subvermispora
produces fatty aldehydes that enable fungal manganese peroxidases to degrade recalcitrant lignin structures
Authors: Kapich Alexander N. , Suzuki Hideki , Hirth Kolby C. , Fernández-Fueyo Elena , Martínez Angel T. , Houtman Carl J. , Hammel Kenneth E. 
Published: March 2024

ABSTRACT

The ability of some white rot basidiomycetes to remove lignin selectively from wood indicates that low molecular weight oxidants have a role in ligninolysis. These oxidants are likely free radicals generated by fungal peroxidases from compounds in the biodegrading wood. Past work supports a role for manganese peroxidases (MnPs) in the production of ligninolytic oxidants from fungal membrane lipids. However, the fatty acid alkylperoxyl radicals initially formed during this process are not reactive enough to attack the major structures in lignin. Here, we evaluate the hypothesis that the peroxidation of fatty aldehydes might provide a source of more reactive acylperoxyl radicals. We found that
Gelatoporia subvermispora
produced
trans-
2-nonenal,
trans-2-
octenal, and n-hexanal (a likely metabolite of
trans-
2,4-decadienal) during the incipient decay of aspen wood. Fungal fatty aldehydes supported the
in vitro
oxidation by MnPs of a nonphenolic lignin model dimer, and also of the monomeric model veratryl alcohol. Experiments with the latter compound showed that the reactions were partially inhibited by oxalate, the chelator that white rot fungi employ to detach Mn
3+
from the MnP active site, but nevertheless proceeded at its physiological concentration of 1 mM. The addition of catalase was inhibitory, which suggests that the standard MnP catalytic cycle is involved in the oxidation of aldehydes. MnP oxidized
trans-
2-nonenal quantitatively to
trans-
2-nonenoic acid with the consumption of one O
2
equivalent. The data suggest that when Mn
3+
remains associated with MnP, it can oxidize aldehydes to their acyl radicals, and the latter subsequently add O
2
to become ligninolytic acylperoxyl radicals.

IMPORTANCE
The biodegradation of lignin by white rot fungi is essential for the natural recycling of plant biomass and has useful applications in lignocellulose bioprocessing. Although fungal peroxidases have a key role in ligninolysis, past work indicates that biodegradation is initiated by smaller, as yet unidentified oxidants that can infiltrate the substrate. Here, we present evidence that the peroxidase-catalyzed oxidation of naturally occurring fungal aldehydes may provide a source of ligninolytic free radical oxidants.
Testing for SARS-CoV-2: lessons learned and current use cases
Authors: Theel Elitza S. , Kirby James E. , Pollock Nira R. 
Published: March 2024

SUMMARY
The emergence and worldwide dissemination of SARS-CoV-2 required both urgent development of new diagnostic tests and expansion of diagnostic testing capacity on an unprecedented scale. The rapid evolution of technologies that allowed testing to move out of traditional laboratories and into point-of-care testing centers and the home transformed the diagnostic landscape. Four years later, with the end of the formal public health emergency but continued global circulation of the virus, it is important to take a fresh look at available SARS-CoV-2 testing technologies and consider how they should be used going forward. This review considers current use case scenarios for SARS-CoV-2 antigen, nucleic acid amplification, and immunologic tests, incorporating the latest evidence for analytical/clinical performance characteristics and advantages/limitations for each test type to inform current debates about how tests should or should not be used.
Corrected and republished from: “Extracellular Vesicle Formation in
Cryptococcus deuterogattii
Impacts Fungal Virulence”
Authors: Castelli Rafael F. , Pereira Alana , Honorato Leandro , Valdez Alessandro , de Oliveira Haroldo C. , Bazioli Jaqueline M. , Garcia Ane W. A. , Klimeck Tabata D'Maiella Freitas , Reis Flavia C. G. , Camillo-Andrade Amanda C. , Santos Marlon D. M. , Carvalho Paulo C. , Zaragoza Oscar , Staats Charley C. , Nimrichter Leonardo , Fill Taícia P. , Rodrigues Marcio L. 
Published: March 2024

ABSTRACT

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known.
NOP16
is a eukaryotic gene that is required for the activity of benzimidazoles against
Cryptococcus deuterogattii
. In this study, during the phenotypic characterization of
C. deuterogattii
mutants expected to lack
NOP16
expression, we observed a reduced EV production. Whole-genome sequencing, RNA-Seq, and cellular proteomics revealed that, contrary to our initial findings, these mutants expressed Nop16 but exhibited altered expression of 14 genes potentially involved in sugar transport. Based on this observation, we designated these mutant strains as Past1 and Past2, representing
p
otentially
a
ltered
s
ugar
t
ransport. Analysis of the small molecule composition of EVs produced by wild-type cells and the Past1 and Past2 mutant strains revealed not only a reduced number of EVs but also an altered small molecule composition. In a
Galleria mellonella
model of infection, the Past1 and Past2 mutant strains were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the mutant cells in
G. mellonella
. These results connect EV biogenesis, cargo, and cryptococcal virulence.
Single-domain antibodies reveal unique borrelicidal epitopes on the Lyme disease vaccine antigen, outer surface protein A (OspA)
Authors: Vance David J. , Basir Saiful , Piazza Carol Lyn , Willsey Graham G. , Haque H. M. Emranul , Tremblay Jacque M. , Rudolph Michael J. , Muriuki Beatrice , Cavacini Lisa , Weis David D. , Shoemaker Charles B. , Mantis Nicholas J. 
Published: March 2024

ABSTRACT

Camelid-derived, single-domain antibodies (V
H
Hs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed V
H
H library directed against the candidate Lyme disease vaccine antigen, outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 from
Borrelia burgdorferi sensu stricto
strain B31, in combination with the canine vaccine RECOMBITEK Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment (“panning”) against recombinant OspA, yielding 21 unique V
H
Hs within two epitope bins, as determined through competition enzyme linked immunosorbent assays (ELISAs) with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry. Six of the monovalent V
H
Hs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, including
B. burgdorferi
agglutination, outer membrane damage, and complement-dependent borreliacidal activity. The V
H
Hs displayed unique reactivity profiles with the seven OspA serotypes associated with
B. burgdorferi
genospecies in the United States and Europe consistent with there being unique epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines.
Salmonella
Enteritidis antitoxin DinJ inhibits NLRP3-dependent canonical inflammasome activation in macrophages
Authors: Gu Dan , Li Ang , Zang Xirui , Huang Tingting , Guo Yaxin , Jiao Xinan , Pan Zhiming 
Published: March 2024

ABSTRACT

The inflammasome is a pivotal component of the innate immune system, acting as a multiprotein complex that plays an essential role in detecting and responding to microbial infections.
Salmonella
Enteritidis have evolved multiple mechanisms to regulate inflammasome activation and evade host immune system clearance. Through screening
S
. Enteritidis C50336Δ
fliC
transposon mutant library, we found that the insertion mutant of
dinJ
increased inflammasome activation. In this study, we demonstrated the genetic connection between the antitoxin DinJ and the toxin YafQ in
S
. Enteritidis, confirming their co-transcription. The deletion mutant Δ
fliC
Δ
dinJ
increased cell death and IL-1β secretion in J774A.1 cells. Western blotting analysis further showed elevated cleaved Caspase-1 product (p10 subunits) and IL-1β secretion in cells infected with Δ
fliC
Δ
dinJ
compared to cells infected with Δ
fliC
. DinJ was found to inhibit canonical inflammasome activation using primary bone marrow-derived macrophages (BMDMs) from
Casp
-/-
C57BL/6 mice. Furthermore, DinJ specifically inhibited NLRP3 inflammasome activation, as demonstrated in BMDMs from
Nlrp3

-/-

and
Nlrc4

-/-

mice. Fluorescence resonance energy transfer (FRET) experiments confirmed the translocation of DinJ into host cells during infection. Finally, we revealed that DinJ could inhibit the secretion of IL-1β and IL-18
in vivo
, contributing to
S
. Enteritidis evading host immune clearance. In summary, our findings provide insights into the role of DinJ in modulating the inflammasome response during
S
. Enteritidis infection, highlighting its impact on inhibiting inflammasome activation and immune evasion.
Distinct dynamics and proximity networks of hub proteins at the prey-invading cell pole in a predatory bacterium
Authors: Remy Ophélie , Santin Yoann G. , Jonckheere Veronique , Tesseur Coralie , Kaljević Jovana , Van Damme Petra , Laloux Géraldine 
Published: March 2024

ABSTRACT

In bacteria, cell poles function as subcellular compartments where proteins localize during specific lifecycle stages, orchestrated by polar “hub” proteins. Whereas most described bacteria inherit an “old” pole from the mother cell and a “new” pole from cell division, generating cell asymmetry at birth, non-binary division poses challenges for establishing cell polarity, particularly for daughter cells inheriting only new poles. We investigated polarity dynamics in the obligate predatory bacterium
Bdellovibrio bacteriovorus
, proliferating through filamentous growth followed by non-binary division within prey bacteria. Monitoring the subcellular localization of two proteins known as polar hubs in other species, RomR and DivIVA, revealed RomR as an early polarity marker in
B. bacteriovorus
. RomR already marks the future anterior poles of the progeny during the predator’s growth phase, during a precise period closely following the onset of divisome assembly and the end of chromosome segregation. In contrast to RomR’s stable unipolar localization in the progeny, DivIVA exhibits a dynamic pole-to-pole localization. This behavior changes shortly before the division of the elongated predator cell, where DivIVA accumulates at all septa and both poles.
In vivo
protein interaction networks for DivIVA and RomR, mapped through endogenous miniTurbo-based proximity labeling, further underscore their distinct roles in cell polarization and reinforce the importance of the anterior “invasive” cell pole in prey-predator interactions. Our work also emphasizes the precise spatiotemporal order of cellular processes underlying
B. bacteriovorus
proliferation, offering insights into the subcellular organization of bacteria with filamentous growth and non-binary division.

IMPORTANCE

In bacteria, cell poles are crucial areas where “hub” proteins orchestrate lifecycle events through interactions with multiple partners at specific times. While most bacteria exhibit one “old” and one “new” pole, inherited from the previous division event, setting polar identity poses challenges in bacteria with non-binary division. This study explores polar proteins in the predatory bacterium
Bdellovibrio bacteriovorus
, which undergoes filamentous growth followed by non-binary division inside another bacterium. Our research reveals distinct localization dynamics of the polar proteins RomR and DivIVA, highlighting RomR as an early “hub” marking polar identity in the filamentous mother cell. Using miniTurbo-based proximity labeling, we uncovered their unique protein networks. Overall, our work provides new insights into the cell polarity in non-binary dividing bacteria.
Peeling the onion: additional layers of regulation in the acid stress response
Authors: Kenney Linda J. 
Published: March 2024

ABSTRACT

Bacteria are capable of withstanding large changes in osmolality and cytoplasmic pH, unlike eukaryotes that tightly regulate their pH and cellular composition. Previous studies on the bacterial acid stress response described a rapid, brief acidification, followed by immediate recovery. More recent experiments with better pH probes have imaged single living cells, and we now appreciate that following acid stress, bacteria maintain an acidic cytoplasm for as long as the stress remains. This acidification enables pathogens to sense a host environment and turn on their virulence programs, for example, enabling survival and replication within acidic vacuoles. Single-cell analysis identified an intracellular pH threshold of ~6.5. Acid stress reduces the internal pH below this threshold, triggering the assembly of a type III secretion system in
Salmonella
and the secretion of virulence factors in the host. These pathways are significant because preventing intracellular acidification of
Salmonella
renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. Although we refer to the acid stress response as singular, it is actually a complex response that involves numerous two-component signaling systems, several amino acid decarboxylation systems, as well as cellular buffering systems and electron transport chain components, among others. In a recent paper in the
Journal of Bacteriology
, M. G. Gorelik, H. Yakhnin, A. Pannuri, A. C. Walker, C. Pourciau, D. Czyz, T. Romeo, and P. Babitzke (J Bacteriol 206:e00354-23, 2024,
https://doi.org/10.1128/jb.00354-23
) describe a new connection linking the carbon storage regulator CsrA to the acid stress response, highlighting new additional layers of complexity.
The intrinsically disordered linker in the single-stranded DNA-binding protein influences DNA replication restart and recombination pathways in
Escherichia coli
K-12
Authors: Sandler Steven J. , Bonde Nina J. , Wood Elizabeth A. , Cox Michael M. , Keck James L. 
Published: March 2024

ABSTRACT

Tetrameric single-stranded (ss) DNA-binding proteins (SSBs) stabilize ssDNA intermediates formed during genome maintenance reactions in
Bacteria
. SSBs also recruit proteins important for these processes through direct SSB-protein interactions, including proteins involved in DNA replication restart and recombination processes. SSBs are composed of an N-terminal oligomerization and ssDNA-binding domain, a C-terminal acidic tip that mediates SSB-protein interactions, and an internal intrinsically disordered linker (IDL). Deletions and insertions into the IDL are well tolerated with few phenotypes, although the largest deletions and insertions exhibit some sensitivity to DNA-damaging agents. To define specific DNA metabolism processes dependent on IDL length,
ssb
mutants that lack 16, 26, 37, or 47 residues of the 57-residue IDL were tested for synthetic phenotypes with mutations in DNA replication restart or recombination genes. We also tested the impact of integrating a fluorescent domain within the SSB IDL using an
ssb::mTur2
insertion mutation. Only the largest deletion tested or the insertion mutation causes sensitivity in any of the pathways. Mutations in two replication restart pathways (PriA-B
1
and PriA-C) showed synthetic lethalities or small colony phenotypes with the largest deletion or insertion mutations. Recombination gene mutations
del(recBCD
) and
del(ruvABC
) show synthetic phenotypes only when combined with the largest
ssb
deletion. These results suggest that a minimum IDL length is important in some genome maintenance reactions in
Escherichia coli
. These include pathways involving PriA-PriB
1
, PriA-PriC, RecFOR, and RecG. The mTur2 insertion in the IDL may also affect SSB interactions in some processes, particularly the PriA-PriB
1
and PriA-PriC replication restart pathways.

IMPORTANCE

ssb
is essential in
Escherichia coli
due to its roles in protecting ssDNA and coordinating genome maintenance events. While the DNA-binding core and acidic tip have well-characterized functions, the purpose of the intrinsically disordered linker (IDL) is poorly understood.
In vitro
studies have revealed that the IDL is important for cooperative ssDNA binding and phase separation. However, single-stranded (ss) DNA-binding protein (SSB) variants with large deletions and insertions in the IDL support normal cell growth. We find that the PriA-PriB
1
and PriA-C replication restart, as well as the RecFOR- and RecG-dependent recombination, pathways are sensitive to IDL length. This suggests that cooperativity, phase separation, or a longer spacer between the core and acidic tip of SSB may be important for specific cellular functions.
Development of a sensor for disulfide bond formation in diverse bacteria
Authors: Dyotima  , Abulaila Sally , Mendoza Jocelyne , Landeta Cristina 
Published: March 2024

ABSTRACT

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. In
Escherichia coli
, disulfide bond formation is catalyzed by DsbA and DsbB enzymes. DsbA is a periplasmic protein that catalyzes disulfide bond formation in substrate proteins, while DsbB is an inner membrane protein that transfers electrons from DsbA to quinones, thereby regenerating the DsbA active state. Actinobacteria including mycobacteria use an alternative enzyme named VKOR, which performs the same function as DsbB. Disulfide bond formation enzymes, DsbA and DsbB/VKOR, represent novel drug targets because their inhibition could simultaneously affect the folding of several cell envelope proteins including virulence factors, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. We have previously developed a cell-based and target-based assay to identify molecules that inhibit the DsbB and VKOR in pathogenic bacteria, using
E. coli
cells expressing a periplasmic β-Galactosidase sensor (β-Gal
dbs
), which is only active when disulfide bond formation is inhibited. Here, we report the construction of plasmids that allows fine-tuning of the expression of the β-Gal
dbs
sensor and can be mobilized into other gram-negative organisms. As an example, when expressed in
Pseudomonas aeruginosa
UCBPP-PA14, which harbors two DsbB homologs, β-Gal
dbs
behaves similarly as in
E. coli
, and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these β-Gal
dbs
reporter plasmids provide a basis to identify novel inhibitors of DsbA and DsbB/VKOR in multidrug-resistant gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria.

IMPORTANCE
Disulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria because inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens.
Oligomeric assembly of the C-terminal and transmembrane region of SARS-CoV-2 nsp3
Authors: Babot Marion , Boulard Yves , Agouda Samira , Pieri Laura , Fieulaine Sonia , Bressanelli Stéphane , Gervais Virginie 
Published: March 2024

ABSTRACT

As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted purified nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negative-stain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFold-predicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA
in vivo
.

IMPORTANCE
Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies.
Human norovirus cultivation systems and their use in antiviral research
Authors: Hayashi Tsuyoshi , Kobayashi Sakura , Hirano Junki , Murakami Kosuke 
Published: March 2024

ABSTRACT

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases, affecting all age groups. Despite its clinical needs, no approved antiviral therapies are available. Since the discovery of HuNoV in 1972, studies on anti-norovirals, mechanism of HuNoV infection, viral inactivation, etc., have been hampered by the lack of a robust laboratory-based cultivation system for HuNoV. A recent breakthrough in the development of HuNoV cultivation systems has opened opportunities for researchers to investigate HuNoV biology in the context of
de novo
HuNoV infections. A tissue stem cell-derived human intestinal organoid/enteroid (HIO) culture system is one of those that supports HuNoV replication reproducibly and, to our knowledge, is most widely distributed to laboratories worldwide to study HuNoV and develop therapeutic strategies. This review summarizes recently developed HuNoV cultivation systems, including HIO, and their use in antiviral studies.