The bacterial flagellum is an appendage structure that provides
a means for motility to promote survival in fluctuating
environments. For the intracellular pathogen Salmonella enterica
serovar Typhimurium to survive within macrophages,
flagellar gene expression must be tightly regulated, and thus,
is controlled at multiple levels, including DNA recombination,
transcription, post-transcription, protein synthesis, and
assembly within host cells. To understand the contribution of
flagella to Salmonella pathogenesis within the host, it is critical
to detect flagella production within macrophages via
microscopy. In this paper, we describe two methods for detecting
bacterial flagella by microscopy both in vitro and in
vivo infection models.
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sulfate-reducing bacterial strain IOR2T was isolated from
a newly found deep-sea hydrothermal vent (OVF, Onnuri
Vent Field) area in the central Indian Ocean ridge (11°2488
S 66°2542E, 2021 m water depth). The 16S rRNA gene sequence
analysis revealed that the strain IOR2T was most closely
related to Desulfovibrio senegalensis BLaC1T (96.7%).
However, it showed low similarity with the members of the
family Desulfovibrionaceae, such as Desulfovibrio tunisiensis
RB22T (94.0%), D. brasiliensis LVform1T (93.9%), D. halophilus
DSM 5663T (93.7%), and Pseudodesulfovibrio aespoeensis
Aspo-2T (93.2%). The strain IOR2T could grow at 23–
42°C (optimum 37°C), pH 5.0–8.0 (optimum pH 7.0) and
with 0.5–6.5% (optimum 3.0%) NaCl. The strain could use
lactate, pyruvate, H2, and glycerol as electron donors and sulfate,
thiosulfate, and sulfite as electron acceptors. The major
fatty acids of the strain IOR2T were iso-C15:0, iso-C17:0, anteiso-
C15:0, and summed feature 9 (C16:0 methyl/iso-C17:1ω9c).
Both the strains IOR2T and BLaC1T could grow with CO2 and
H2 as the sole sources of carbon and energy, respectively. Genomic
evidence for the Wood-Ljungdahl pathway in both
the strains reflects chemolithoautotrophic growth. The DNA
G + C content of the strain IOR2T and BLaC1T was 58.1–60.5
mol%. Based on the results of the phylogenetic and physiologic
studies, Paradesulfovibrio onnuriensis gen. nov., sp.
nov. with the type strain IOR2T (= KCTC 15845T = MCCC
1K04559T) was proposed to be a member of the family Desulfovibrionaceae.
We have also proposed the reclassification
of D. senegalensis as Paradesulfovibrio senegalensis comb. nov.
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A strictly anaerobic, dissimilatory Fe(III)-reducing hyperthermophilic
archaeon, designated as strain IOH1T, was isolated
from a new deep-sea hydrothermal vent (Onnuri Vent Field)
area in the Central Indian Ocean ridge. Strain IOH1T showed
> 99% 16S rRNA gene sequence similarity with Thermococcus
celericrescens TS2T (99.4%) and T. siculi DSM 12349T (99.2%).
Additional three species T. barossii SHCK-94T (99.0%), T. celer
Vu13T (98.8%), and T. piezophilus (98.6%) showed > 98.6%
of 16S rRNA gene sequence similarity, however, the maximum
OrthoANI value is 89.8% for the genome of T. celericrescens
TS2T. Strain IOH1T cells are coccoid, 1.2–1.8 μm
in diameter, and motile by flagella. Growth was at 70–82°C
(optimum 80°C), pH 5.4–8.0 (optimum pH 6.0) with 2–4%
(optimum 3%) NaCl. Growth of strain IOH1T was enhanced
by starch, pyruvate, D(+)-maltose and maltodextrin as a carbon
sources, and elemental sulfur as an electron acceptor;
clearly different from those of related species T. celecrescens
DSM 17994T and T. siculi DSM 12349T. Strain IOH1T, T. celercrescence
DSM 17994T, and T. siculi DSM 12349T reduced
soluble Fe(III)-citrate present in the medium, whereas the
amount of total cellular proteins increased with the concomitant
accumulation of Fe(II). We determined a circular chromosome
of 2,234 kb with an extra-chromosomal archaeal
plasmid, pTI1, of 7.7 kb and predicted 2,425 genes. The DNA
G + C content was 54.9 mol%. Based on physiological properties,
phylogenetic, and genome analysis, we proposed that
strain IOH1T (= KCTC 15844T = JCM 39077T) is assigned to
a new species in the genus Thermococcus and named Thermococcus
indicus sp. nov.
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We measured the grazing and growth response of the mixotrophic
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represented different pigment types (phycoerythrin-
rich, PE, and phycocyanin-rich, PC) and phylogenetic
clusters. The grazing experiments were conducted with laboratory
cultures acclimated to 10 μmol photon/m2/sec (low
light, LL) and 100 μmol photon/m2/sec (moderate light, ML),
either in the dark or at four different irradiances ranging from
low (6 μmol photon/m2/sec) to high (1,500 μmol photon/m2/
sec) light intensity. Poterioochromonas malhamensis preferred
the larger, green PC-rich picocyanobacteria to the smaller,
red PE-rich picocyanobacterial, and heterotrophic bacteria.
The feeding and growth rates of P. malhamensis were sensitive
to the actual light conditions during the experiments;
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LL conditions than at high light intensity. In summary, our results found strain-specific ingestion and growth rates of
the flagellate; an effect of the preculturing conditions, and,
unexpectedly, a direct adverse effect of high light levels. We
conclude that this flagellate may avoid exposure to high surface
light intensities commonly encountered in temperate
lakes during the summer.
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composite design. To obtain higher survivability in mortar,
the sporulation process is essential, and additional factors
such as Mn2+, Fe2+, and Ca2+ were found to contribute
to sporulation. A mixture of L. boronitolerans YS11 spore
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in 7 days. Furthermore, calcium carbonate precipitation was
observed over the crack surface. Thus, we confirmed that mortar
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analyzed bacilysin-nonproducing strain OGU1 which was
obtained by bacA-targeted pMutin T3 insertion into the
parental strain genome resulting in a genomic organization
(bacA::lacZ::erm::bacABCDEF) to form an IPTG-inducible
bac operon. Although IPTG induction provided 3- to 5-fold
increment in the transcription of bac operon genes, no bacilysin
activity was detectable in bioassays and inability of the
OGU1 to form bacilysin was confirmed by UPLC-mass spectrometry
analysis. Phenotypic analyses revealed the deficiencies
in OGU1 with respect to colony pigmentation, spore coat
proteins, spore resistance and germination, which could be
rescued by external addition of bacilysin concentrate into its
cultures. 2DE MALDI-TOF/MS and nanoLC-MS/MS were
used as complementary approaches to compare cytosolic proteomes
of OGU1. 2-DE identified 159 differentially expressed
proteins corresponding to 121 distinct ORFs. In nanoLCMS/
MS, 76 proteins were differentially expressed in OGU1.
Quantitative transcript analyses of selected genes validated
the proteomic findings. Overall, the results pointed to the impact
of bacilysin on expression of certain proteins of sporulation
and morphogenesis; the members of mother cell compartment-
specific σE and σK regulons in particular, quorum
sensing and two component-global regulatory systems, peptide
transport, stress response as well as CodY- and ScoCregulated
proteins.
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Apical periodontitis is caused by biofilm-mediated root canal
infection. Early phase oral bacterial biofilms are inhibited by
Lactobacillus plantarum lipoteichoic acid (Lp.LTA). However,
mature biofilms that develop over 3 weeks are more resistant
to traditional endodontic medicaments. Therefore, this study
examined the effectiveness of Lp.LTA on disrupting mature
Enterococcus faecalis biofilms, and on enhancing the effects
of endodontic medicaments. LTA was purified from L. plantarum
through butanol extraction followed by hydrophobic
and ion-exchange chromatography. E. faecalis biofilms were
formed over 3 weeks on glass bottom dishes and in dentin
blocks obtained from human single-rooted premolars. These
mature biofilms were treated with or without Lp.LTA for 1 h,
followed by additional treatment with either chlorhexidine
digluconate (CHX), calcium hydroxide (CH), or triple antibiotics
for 24 h. Biofilms on glass were live/dead stained and
quantified by ZEN through confocal laser microscopy. Biofilms
in dentin were fixed, sputter coated and analyzed by
ImageJ with scanning electron microscopy. Preformed E. faecalis
mature biofilms on the culture dishes were dose-dependently
disrupted by Lp.LTA. Lp.LTA potentiated the effects
of CHX or CH on the disruption of mature biofilm. Interestingly,
CHX-induced disruption of preformed E. faecalis
mature biofilms was synergistically enhanced only when pretreated
with Lp.LTA. Furthermore, in the dentin block model,
Lp.LTA alone reduced E. faecalis mature biofilm and
pre-treatment with Lp.LTA promoted the anti-biofilm activity
of CHX. Lp.LTA could be an anti-biofilm or supplementary
agent that can be effective for E. faecalis-biofilminduced
diseases.
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Bovine mastitis is a common disease in the dairy industry
that causes great economic losses. As the primary pathogen
of contagious mastitis, Staphylococcus aureus (S. aureus) can
invade bovine mammary epithelial cells, thus evading immune
defenses and resulting in persistent infection. Recently,
autophagy has been considered an important mechanism for
host cells to clear intracellular pathogens. In the current study,
autophagy caused by S. aureus was detected, and the correlation
between autophagy and intracellular S. aureus survival
was assessed. First, a model of intracellular S. aureus infection
was established. Then, the autophagy of MAC-T cells was
evaluated by confocal microscopy and western blot. Moreover,
the activation of the PI3K-Akt-mTOR and ERK1/2 signaling
pathways was determined by western blot. Finally, the
relationship between intracellular bacteria and autophagy
was analyzed by using autophagy regulators (3-methyladenine
[3-MA], rapamycin [Rapa] and chloroquine [CQ]). The results showed that S. aureus caused obvious induction of
autophagosome formation, transformation of LC3I/II, and
degradation of p62/SQSTM1 in MAC-T cells; furthermore,
the PI3K-Akt-mTOR and ERK1/2 signaling pathways were
activated. The number of intracellular S. aureus increased
significantly with autophagy activation by rapamycin, whereas
the number decreased when the autophagy flux was inhibited
by chloroquine. Therefore, this study indicated that intracellular
S. aureus can induce autophagy and utilize it to survive
in bovine mammary epithelial cells.
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Academy of Breastfeeding Medicine Clinical Protocol #36: The Mastitis Spectrum, Revised 2022 Katrina B. Mitchell, Helen M. Johnson, Juan Miguel Rodríguez, Anne Eglash, Charlotte Scherzinger, Kyle Widmer, Pamela Berens, Brooke Miller Breastfeeding Medicine.2022; 17(5): 360. CrossRef
Streptococcus agalactiae-induced autophagy of bovine mammary epithelial cell via PI3K/AKT/mTOR pathway Mengzhu Qi, Hao Geng, Na Geng, Yukun Cui, Changxi Qi, Guodong Cheng, Kaimin Song, Liping Hu, Yongxia Liu, Jianzhu Liu, Bo Han Journal of Dairy Research.2022; 89(2): 178. CrossRef
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Streptococcus pneumoniae is a Gram-positive pathogen with
high morbidity and mortality globally but some of its pathogenesis
remains unknown. Previous research has provided
evidence that aminopeptidase N (PepN) is most likely a virulence
factor of S. pneumoniae. However, its role in S. pneumoniae
virulence and its interaction with the host remains
to be confirmed. We generated a pepN gene deficient mutant
strain and found that its virulence for mice was significantly
attenuated as were in vitro adhesion and invasion of host
cells. The PepN protein could induce a strong innate immune
response in vivo and in vitro and induced secretion of IL-6
and TNF-α by primary peritoneal macrophages via the rapid
phosphorylation of MAPK and PI3K/AKT signaling pathways
and this was confirmed using specific pathway inhibitors.
In conclusion, PepN is a novel virulence factor that is
essential for the virulence of S. pneumoniae and induces host
innate immunity via MAPK and PI3K/AKT signaling.
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