bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficacy of aspergillomarasmine A/meropenem combinations with and without avibactam against bacterial strains producing multiple β-lactamases
Authors: Rotondo Caitlyn M. , Wright Gerard D. 
Published: August 2024

ABSTRACT

The effectiveness of β-lactam antibiotics is increasingly threatened by resistant bacteria that harbor hydrolytic β-lactamase enzymes. Depending on the class of β-lactamase present, β-lactam hydrolysis can occur through one of two general molecular mechanisms. Metallo-β-lactamases (MBLs) require active site Zn
2+
ions, whereas serine-β-lactamases (SBLs) deploy a catalytic serine residue. The result in both cases is drug inactivation via the opening of the β-lactam warhead of the antibiotic. MBLs confer resistance to most β-lactams and are non-susceptible to SBL inhibitors, including recently approved diazabicyclooctanes, such as avibactam; consequently, these enzymes represent a growing threat to public health. Aspergillomarasmine A (AMA), a fungal natural product, can rescue the activity of the β-lactam antibiotic meropenem against MBL-expressing bacterial strains. However, the effectiveness of this β-lactam/β-lactamase inhibitor combination against bacteria producing multiple β-lactamases remains unknown. We systematically investigated the efficacy of AMA/meropenem combination therapy with and without avibactam against 10
Escherichia coli
and 10
Klebsiella pneumoniae
laboratory strains tandemly expressing single MBL and SBL enzymes. Cell-based assays demonstrated that laboratory strains producing NDM-1 and KPC-2 carbapenemases were resistant to the AMA/meropenem combination but became drug-susceptible upon adding avibactam. We also probed these combinations against 30 clinical isolates expressing multiple β-lactamases.
E. coli
,
Enterobacter cloacae,
and
K. pneumoniae
clinical isolates were more susceptible to AMA, avibactam, and meropenem than
Pseudomonas aeruginosa
and
Acinetobacter baumannii
isolates. Overall, the results demonstrate that a triple combination of AMA/avibactam/meropenem has potential for empirical treatment of infections caused by multiple β-lactamase-producing bacteria, especially Enterobacterales.
Enhanced accumulation of γ-aminobutyric acid by deletion of aminotransferase genes involved in γ-aminobutyric acid catabolism in engineered
Halomonas elongata
Authors: Zou Ziyan , Kaothien-Nakayama Pulla , Nakayama Hideki 
Published: August 2024

ABSTRACT

Halomonas elongata
OUT30018 is a moderately halophilic bacterium that synthesizes and accumulates ectoine as an osmolyte by activities of the enzymes encoded by the high salinity-inducible
ectABC
operon. Previously, we engineered a γ-aminobutyric acid (GABA)-producing
H. elongata
GOP-Gad (Δ
ectABC::mCherry-HopGadmut
) from an ectoine-deficient mutant of this strain due to its ability to use high-salinity biomass waste as substrate. Here, to further increase GABA accumulation, we deleted
gabT,
which encodes GABA aminotransferase (GABA-AT) that catalyzes the first step of the GABA catabolic pathway, from the
H. elongata
GOP-Gad genome. The resulting strain
H. elongata
ZN3 (Δ
ectABC::mCherry-HopGadmut
Δ
gabT
) accumulated 291 µmol/g cell dry weight (CDW) of GABA in the cells, which is a 1.5-fold increase from
H. elongata
GOP-Gad’s 190 µmol/g CDW. This result has confirmed the role of GABA-AT in the GABA catabolic pathway. However, redundancy in endogenous GABA-AT activity was detected in a growth test, where a
gabT
-deletion mutant of
H. elongata
OUT30018 was cultured in a medium containing GABA as the sole carbon and nitrogen sources. Because L-2,4-diaminobutyric acid aminotransferase (DABA-AT), encoded by an
ectB
gene of the
ectABC
operon, shares sequence similarity with GABA-AT, a complementation analysis of the
gabT
and the
ectB
genes was performed in the
H. elongata
ZN3 genetic background to test the involvement of DABA-AT in the redundancy of GABA-AT activity. Our results indicate that the expression of DABA-AT can restore GABA-AT activity in
H. elongata
ZN3 and establish DABA-AT’s aminotransferase activity toward GABA
in vivo
.

IMPORTANCE

In this study, we were able to increase the yield of GABA by 1.5 times in the GABA-producing
H. elongata
ZN3 strain by deleting the
gabT
gene, which encodes GABA-AT, the initial enzyme of the GABA catabolic pathway. We also report the first
in vivo
evidence for GABA aminotransferase activity of an
ectB
-encoded DABA-AT, confirming a longstanding speculation based on the reported
in vitro
GABA-AT activity of DABA-AT. According to our findings, the DABA-AT enzyme can catalyze the initial step of GABA catabolism, in addition to its known function in ectoine biosynthesis. This creates a cycle that promotes adequate substrate flow between the two pathways, particularly during the early stages of high-salinity stress response when the expression of the
ectB
gene is upregulated.
A promoter–RBS library for fine-tuning gene expression in
Methanosarcina acetivorans
Authors: Zhu Ping , Molina Resendiz Mariana , von Ossowski Ingemar , Scheller Silvan 
Published: August 2024

ABSTRACT

Methanogens are the main biological producers of methane on Earth.
Methanosarcina acetivorans
is one of the best characterized methanogens that has powerful genetic tools for genome editing. To study the physiology of this methanogen in further detail as well as to effectively balance the flux of their engineered metabolic pathways in expansive project undertakings, there is the need for controlled gene expression, which then requires the availability of well-characterized promoters and ribosome-binding sites (RBS). In this study, we constructed a library of 33 promoter–RBS combinations that includes 13 wild-type and 14 hybrid combinations, as well as six combination variants in which the 5'-untranslated region (5'UTR) was rationally engineered. The expression strength for each combination was calculated by inducing the expression of the β-glucuronidase reporter gene in
M. acetivorans
cells in the presence of the two most used growth substrates, either methanol (MeOH) or trimethyl amine (TMA). In this study, the constructed library covers a relatively wide range (140-fold) between the weakest and strongest promoter–RBS combination as well as shows a steady increase and allows different levels of gene expression. Effects on the gene expression strength were also assessed by making measurements at three distinct growth phases for all 33 promoter–RBS combinations. Our promoter–RBS library is effective in enabling the fine-tuning of gene expression in
M. acetivorans
for physiological studies and the design of metabolic engineering projects that, e.g., aim for the biotechnological valorization of one-carbon compounds.

IMPORTANCE

Methanogenic archaea are potent producers of the greenhouse gas methane and thus contribute substantially to global warming. Under controlled conditions, these microbes can catalyze the production of biogas, which is a renewable fuel, and might help counter global warming and its effects. Engineering the primary metabolism of
Methanosarcina acetivorans
to render it better and more useful requires controllable gene expression, yet only a few well-characterized promoters and RBSs are presently available. Our study rectifies this situation by providing a library of 33 different promoter–RBS combinations with a 140-fold dynamic range in expression strength. Future metabolic engineering projects can take advantage of this library by using these promoter–RBS combinations as an efficient and tunable gene expression system for
M. acetivorans
. Furthermore, the methodologies we developed in this study could also be utilized to construct promoter libraries for other types of methanogens.
Exploring strain-level diversity in the gut microbiome through mucin particle adhesion
Authors: Nishiyama Keita , Murakami Ryuta , Nakahata Masaki , Zhou Binghui , Hashikura Nanami , Kaneko Hiroki , Namai Fu , Ikeda-Ohtsubo Wakako , Xiao Jin-Zhong , Kitazawa Haruki , Odamaki Toshitaka 
Published: August 2024

ABSTRACT

Mucin glycoproteins are a significant source of carbon for the gut bacteria. Various gut microbial species possess diverse hydrolytic enzymes and catabolic pathways for breaking down mucin glycans, resulting in competition for the limited nutrients within the gut environment. Adherence to mucin glycans represents a crucial strategy used by gut microbes to access nutrient reservoirs. Understanding these properties is pivotal for comprehending the survival mechanisms of bacteria in the gastrointestinal tract. However, characterization of individual strains within the vast array of coexisting bacteria in the microbiome is challenging. To investigate this, we developed mucin-immobilized particles by immobilizing porcine gastric mucin (PGM) onto glass beads chemically modified with boronic acid. These PGM-immobilized particles were then anaerobically cultured with human fecal microbiota, and the bacteria adhering to PGM were isolated. Interestingly, the microbiome composition remained largely unchanged irrespective of PGM immobilization. Nonetheless, bacteria isolated from PGM-immobilized glass particles exhibited notably higher
N
-acetylgalactosaminidase activity compared to the control beads. Furthermore,
Bacteroides
strains isolated from PGM-immobilized glass particles displayed enhanced adhesive and metabolic properties to PGM. These findings underscore the utility of PGM particles in enriching and isolating specific microbes. Moreover, they highlight substantial differences in microbial properties at the strain level. We anticipate that PGM-immobilized particles will advance culture-based microbiome research, emphasizing the significance of strain-level characterization.

IMPORTANCE
Metabolism of mucin glycans by gut bacteria represents a crucial strategy for accessing nutrient reservoirs. The efficacy of mucin glycan utilization among gut bacteria hinges on the metabolic capabilities of individual strains, necessitating meticulous strain-level characterization. In this investigation, we used glass beads chemically immobilized with mucins to selectively enrich bacteria from fecal fermentation cultures, based on their superior adhesion to and metabolism of mucin glycoproteins. These findings lend support to the hypothesis that the physical interactions between bacteria and mucin glycoprotein components directly correlate with their capacity to utilize mucins as nutrient sources. Furthermore, our study implies that physical proximity may significantly influence bacterial nutrient acquisition within the ecosystem, facilitating gut bacteria’s access to carbohydrate components.
Mycoplasma hyopneumoniae
inhibits the unfolded protein response to prevent host macrophage apoptosis and M2 polarization
Authors: Liu Tong , Zhang Yujuan , Zhao Huanjun , Wu Qi , Xin Jiuqing , Pan Qiao 
Published: August 2024

ABSTRACT

Enzootic pneumonia caused by
Mycoplasma hyopneumoniae
(
M. hyopneumoniae
) has inflicted substantial economic losses on the global pig industry. The progression of
M. hyopneumoniae
induced-pneumonia is associated with lung immune cell infiltration and extensive proinflammatory cytokine secretion. Our previous study established that
M. hyopneumoniae
disrupts the host unfolded protein response (UPR), a process vital for the survival and immune function of macrophages. In this study, we demonstrated that
M. hyopneumoniae
targets the UPR- and caspase-12-mediated endoplasmic reticulum (ER)-associated classical intrinsic apoptotic pathway to interfere with host cell apoptosis signaling, thereby preserving the survival of host tracheal epithelial cells (PTECs) and alveolar macrophages (PAMs) during the early stages of infection. Even in the presence of apoptosis inducers, host cells infected with
M. hyopneumoniae
exhibited an anti-apoptotic potential. Further analyses revealed that
M. hyopneumoniae
suppresses the three UPR branches and their induced apoptosis. Interestingly, while UPR activation typically drives host macrophages toward an M2 polarization phenotype,
M. hyopneumoniae
specifically obstructs this process to maintain a proinflammatory phenotype in the host macrophages. Overall, our findings propose that
M. hyopneumoniae
inhibits the host UPR to sustain macrophage survival and a proinflammatory phenotype, which may be implicated in its pathogenesis in inducing host pneumonia.
The host GTPase Dynamin 2 modulates apical junction structure to control cell-to-cell spread of
Listeria monocytogenes
Authors: Tijoriwalla Serena , Liyanage Thiloma , Herath Thilina U. B. , Lee Nicole , Rehman Attika , Gianfelice Antonella , Ireton Keith 
Published: August 2024

ABSTRACT

The food-borne pathogen
Listeria monocytogenes
uses actin-based motility to generate plasma membrane protrusions that mediate the spread of bacteria between host cells. In polarized epithelial cells, efficient protrusion formation by
L. monocytogenes
requires the secreted bacterial protein InlC, which binds to a carboxyl-terminal Src homology 3 (SH3) domain in the human scaffolding protein Tuba. This interaction antagonizes Tuba, thereby diminishing cortical tension at the apical junctional complex and enhancing
L. monocytogenes
protrusion formation and spread. Tuba contains five SH3 domains apart from the domain that interacts with InlC. Here, we show that human GTPase Dynamin 2 associates with two SH3 domains in the amino-terminus of Tuba and acts together with this scaffolding protein to control the spread of
L. monocytogenes
. Genetic or pharmacological inhibition of Dynamin 2 or knockdown of Tuba each restored normal protrusion formation and spread to a bacterial strain deleted for the
inlC
gene (∆
inlC
). Dynamin 2 localized to apical junctions in uninfected human cells and protrusions in cells infected with
L. monocytogenes
. Localization of Dynamin 2 to junctions and protrusions depended on Tuba. Knockdown of Dynamin 2 or Tuba diminished junctional linearity, indicating a role for these proteins in controlling cortical tension. Infection with
L. monocytogenes
induced InlC-dependent displacement of Dynamin 2 from junctions, suggesting a possible mechanism of antagonism of this GTPase. Collectively, our results show that Dynamin 2 cooperates with Tuba to promote intercellular tension that restricts the spread of ∆
inlC Listeria
. By expressing InlC, wild-type
L. monocytogenes
overcomes this restriction.
The impact of butyrate on group B
Streptococcus
-induced intestinal barrier disruption
Authors: Dominguez Kristen , Pearah Alexia N. , Lindon April K. , Worthington Leigh-Anne M. , Carter Rico R. , John-Lewis Edwards Nichol , Ho Thao T. B. , Darch Sophie E. , Randis Tara M. 
Published: August 2024

ABSTRACT

Group B Streptococcus (
Streptococcus agalactiae
; GBS) is a leading cause of neonatal sepsis worldwide. As a pathobiont of the intestinal tract, it is capable of translocating across barriers leading to invasive disease. Neonatal susceptibility to invasive disease stems from immature intestinal barriers. GBS intestinal colonization induces major transcriptomic changes in the intestinal epithelium related to barrier function. Butyrate, a microbial metabolite produced by fermentation of dietary fiber, bolsters intestinal barrier function against enteric pathogens, and these effects can be transferred
in utero
via the placenta to the developing fetus. Our aim was to determine if butyrate mitigates GBS disruption of intestinal barriers. We used human intestinal epithelial cell (IEC) lines to evaluate the impact of butyrate on GBS-induced cell death and GBS adhesion and invasion. IECs and human fetal tissue-derived enteroids were used to evaluate monolayer permeability. We evaluated the impact of maternal butyrate treatment (mButyrate) using our established mouse model of neonatal GBS intestinal colonization and late-onset sepsis. We found that butyrate reduces GBS-induced cell death, GBS invasion, monolayer permeability, and translocation
in vitro
. In mice, mButyrate decreases GBS intestinal burden in offspring. Our results demonstrate the importance of bacterial metabolites, such as butyrate, in their potential to bolster epithelial barrier function and mitigate neonatal sepsis risk.

IMPORTANCE

Group B
Streptococcus
(GBS) is a leading cause of neonatal morbidity and mortality. It is a commensal of the intestines that can translocate across barriers leading to sepsis in vulnerable newborns. With the rise in antibiotic-resistant strains and no licensed vaccine, there is an urgent need for preventative strategies. Butyrate, a short-chain fatty acid metabolized in the gut, enhances barrier function against pathogens. Importantly, butyrate is transferred
in utero
, conferring these benefits to infants. Here, we demonstrate that butyrate reduces GBS colonization and epithelial invasion. These effects were not microbiome-driven, suggesting butyrate directly impacts epithelial barrier function. Our results highlight the potential impact of maternal dietary metabolites, like butyrate, as a strategy to mitigate neonatal sepsis risk.
The microbiome-derived antibacterial lugdunin acts as a cation ionophore in synergy with host peptides
Authors: Berscheid Anne , Straetener Jan , Schilling Nadine A. , Ruppelt Dominik , Konnerth Martin C. , Schittek Birgit , Krismer Bernhard , Peschel Andreas , Steinem Claudia , Grond Stephanie , Brötz-Oesterhelt Heike 
Published: August 2024

ABSTRACT

Lugdunin is a microbiome-derived antibacterial agent with good activity against Gram-positive pathogens
in vitro
and in animal models of nose colonization and skin infection. We have previously shown that lugdunin depletes bacterial energy resources by dissipating the membrane potential of
Staphylococcus aureus
. Here, we explored the mechanism of action of lugdunin in more detail and show that lugdunin quickly depolarizes cytoplasmic membranes of different bacterial species and acidifies the cytoplasm of
S. aureus
within minutes due to protonophore activity. Varying the salt species and concentrations in buffers revealed that not only protons are transported, and we demonstrate the binding of the monovalent cations K
+
, Na
+
, and Li
+
to lugdunin. By comparing known ionophores with various ion transport mechanisms, we conclude that the ion selectivity of lugdunin largely resembles that of 15-mer linear peptide gramicidin A. Direct interference with the main bacterial metabolic pathways including DNA, RNA, protein, and cell wall biosyntheses can be excluded. The previously observed synergism of lugdunin with dermcidin-derived peptides such as DCD-1 in killing
S. aureus
is mechanistically based on potentiated membrane depolarization. We also found that lugdunin was active against certain eukaryotic cells, however strongly depending on the cell line and growth conditions. While adherent lung epithelial cell lines were almost unaffected, more sensitive cells showed dissipation of the mitochondrial membrane potential. Lugdunin seems specifically adapted to its natural environment in the respiratory tract. The ionophore mechanism is refractory to resistance development and benefits from synergy with host-derived antimicrobial peptides.

IMPORTANCE

The vast majority of antimicrobial peptides produced by members of the microbiome target the bacterial cell envelope by many different mechanisms. These compounds and their producers have evolved side-by-side with their host and were constantly challenged by the host’s immune system. These molecules are optimized to be well tolerated at their physiological site of production, and their modes of action have proven efficient
in vivo
. Imbalancing the cellular ion homeostasis is a prominent mechanism among antibacterial natural products. For instance, over 120 naturally occurring polyether ionophores are known to date, and antimicrobial peptides with ionophore activity have also been detected in microbiomes. In this study, we elucidated the mechanism underlying the membrane potential-dissipating activity of the thiazolidine‐containing cycloheptapeptide lugdunin, the first member of the fibupeptides discovered in a commensal bacterium from the human nose, which is a promising future probiotic candidate that is not prone to resistance development.
Age and sex specific thresholds for risk stratification of cardiovascular disease and clinical decision making: prospective open cohort study
Authors: Xu Zhe , Usher-Smith Juliet , Pennells Lisa , Chung Ryan , Arnold Matthew , Kim Lois , Kaptoge Stephen , Sperrin Matthew , Di Angelantonio Emanuele , Wood Angela M 
Published: August 2024

ObjectiveTo quantify the potential advantages of using 10 year risk prediction models for cardiovascular disease, in combination with risk thresholds specific to both age and sex, to identify individuals at high risk of cardiovascular disease for allocation of statin treatment.DesignProspective open cohort study.SettingPrimary care data from the UK Clinical Practice Research Datalink GOLD, linked with hospital admissions from Hospital Episode Statistics and national mortality records from the Office for National Statistics in England, 1 January 2006 to 31 May 2019.Participants1 046 736 individuals (aged 40-85 years) with no cardiovascular disease, diabetes, or a history of statin treatment at baseline using data from electronic health records.Main outcome measures10 year risk of cardiovascular disease, calculated with version 2 of the QRISK cardiovascular disease risk algorithm (QRISK2), with two main strategies to identify individuals at high risk: in strategy A, estimated risk was a fixed cut-off value of ≥10% (ie, as per the UK National Institute for Health and Care Excellence guidelines); in strategy B, estimated risk was ≥10% or ≥90th centile of age and sex specific risk distributions.ResultsCompared with strategy A, strategy B stratified 20 241 (149.8%) more women aged ≤53 years and 9832 (150.2%) more men aged ≤47 years as having a high risk of cardiovascular disease; for all other ages the strategies were the same. Assuming that treatment with statins would be initiated in those identified as high risk, differences in the estimated gain in cardiovascular disease-free life years from statin treatment for strategy B versus strategy A were 0.14 and 0.16 years for women and men aged 40 years, respectively; among individuals aged 40-49 years, the numbers needed to treat to prevent one cardiovascular disease event for strategy B versus strategy A were 39 versus 21 in women and 19 versus 15 in men, respectively.ConclusionsThis study quantified the potential gains in cardiovascular disease-free life years when implementing prevention strategies based on age and sex specific risk thresholds instead of a fixed risk threshold for allocation of statin treatment. Such gains should be weighed against the costs of treating more younger people with statins for longer.