Antibiotic treatment failure threatens our ability to control bacterial infections that can cause chronic diseases. Persister bacteria
are a subpopulation of physiological variants that becomes highly tolerant to antibiotics. Membrane proteins play crucial
roles in all living organisms to regulate cellular physiology. Although a diverse membrane component involved in persistence
can result in antibiotic treatment failure, the regulations of antibiotic persistence by membrane proteins has not been fully
understood. In this review, we summarize the recent advances in our understanding with regards to membrane proteins in
Gram-negative bacteria as a regulator for antibiotic persistence, highlighting various physiological mechanisms in bacteria.
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Three major proteases, elastase B (LasB), protease IV (PIV),
and elastase A (LasA) expressed in Pseudomonas aeruginosa
play important roles in infections and pathogeneses. These
are activated by a proteolytic cascade initiated by the activation
of LasB. In this study, we investigated whether LasB
could be inhibited using its propeptide (LasBpp). Although
LasA and PIV were inhibited by their propeptides, LasB was
not inhibited by purified LasBpp because LasB degraded LasBpp.
To address this problem, mutant LasBpp variants were constructed
to obtain a mutant LasBpp resistant to LasB degradation.
A C-terminal deletion series of LasBpp was tested in
vivo, and two positive candidates, T2 and T2-1, were selected.
However, both caused growth retardation and were unstably
expressed in vivo. Since deleting the C-terminal end of LasBpp
significantly affected its stable expression, substitution mutations
were introduced at the two amino acids near the
truncation site of T2-1. The resulting mutants, LasBppE172D,
LasBppG173A, and LasBppE172DG173A, significantly diminished LasB
activity when overexpressed in vivo and were stably expressed
in MW1, a quorum sensing mutant that does not produce
LasB. In vitro analysis showed that purified LasBppE172DG173A
inhibited LasB activity to a small extent. Summarizing, Cterminal
modification of LasBpp profoundly affected the stable
expression of LasBpp, and little enhanced the ability of
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A critical obstacle to the successful treatment of colorectal
cancer (CRC) is chemoresistance. Chemoresistant CRC cells
contribute to treatment failure by providing a mechanism
of drug lethargy and modifying chemoresistance-associated
molecules. The gut microbiota provide prophylactic and therapeutic
effects by targeting CRC through anticancer mechanisms.
Among them, Lactobacillus plantarum contributes
to the health of the host and is clinically effective in treating
CRC. This study confirmed that 5-fluorouracil (5-FU)-resistant
CRC HCT116 (HCT116/5FUR) cells acquired butyrateinsensitive
properties. To date, the relationship between 5-
FU-resistant CRC and butyrate resistance has not been elucidated.
Here, we demonstrated that the acquisition of butyrate
resistance in HCT116/5FUR cells was strongly correlated
with the inhibition of the expression and function of
SMCT1, a major transporter of butyrate in colonocytes. L.
plantarum-cultured cell-free supernatant (LP) restored the
functional expression of SMCT1 in HCT116/5FUR cells, leading
to butyrate-induced antiproliferative effect and apoptosis.
These results suggest that LP has a synergistic effect on the
SMCT1/butyrate-mediated tumor suppressor function and
is a potential chemosensitizer to overcome dual 5-FU and butyrate
resistance in HCT116 cells.
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Streptococcus gordonii, a Gram-positive commensal bacterium,
is an opportunistic pathogen closely related to initiation
and progression of various oral diseases, such as periodontitis
and dental caries. Its biofilm formation is linked
with the development of such diseases by enhanced resistance
against antimicrobial treatment or host immunity. In the
present study, we investigated the effect of short-chain fatty
acids (SCFAs) on the biofilm formation of S. gordonii. SCFAs,
including sodium acetate (NaA), sodium propionate (NaP),
and sodium butyrate (NaB), showed an effective inhibitory
activity on the biofilm formation of S. gordonii without reduction
in bacterial growth. SCFAs suppressed S. gordonii
biofilm formation at early time points whereas SCFAs did
not affect its preformed biofilm. A quorum-sensing system
mediated by competence-stimulating peptide (CSP) is known
to regulate biofilm formation of streptococci. Interestingly,
SCFAs substantially decreased mRNA expression of comD
and comE, which are CSP-sensor and its response regulator
responsible for CSP pathway, respectively. Although S. gordonii
biofilm formation was enhanced by exogenous synthetic
CSP treatment, such effect was not observed in the
presence of SCFAs. Collectively, these results suggest that
SCFAs have an anti-biofilm activity on S. gordonii through
inhibiting comD and comE expression which results in negative
regulation of CSP quorum-sensing system. SCFAs could
be an effective anti-biofilm agent against S. gordonii for the
prevention of oral diseases.
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by measuring the physical and chemical properties of
cells based on their light scattering behavior and fluorescence.
Compared to molecular or culture-based approaches, flow
cytometry is suitable for the online monitoring of microbial
water quality because of its relatively simple sample preparation
process, rapid analysis time, and high-resolution phenotypic
data. Advanced statistical techniques (e.g., denoising
and binning) can be utilized to successfully calculate phenotypic
diversity by processing the scatter data obtained from
flow cytometry. These phenotypic diversities were well correlated
with taxonomic-based diversity computed using nextgeneration
16S RNA gene sequencing. The protocol provided
in this paper should be a useful guide for a fast and reliable
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such as chronic inflammatory disease, intestinal infection
and colorectal cancer. To identify such dysregulations,
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microorganisms composing the human gut microbiome. In
this study, we used the “microscomics” approach, which consists
of creating an ultrastructural repertoire of all the cell-like
objects composing stool samples from healthy donors using
transmission electron microscopy (TEM). We used TEM to
screen ultrathin sections of 8 resin-embedded stool samples.
After exploring hundreds of micrographs, we managed to
elaborate ultrastructural categories based on morphological
criteria or features. This approach explained many inconsistencies
observed with other techniques, such as metagenomics
and culturomics. We highlighted the value of our cultureindependent
approach by comparing our microscopic images
to those of cultured bacteria and those reported in the
literature. This study helped to detect “minimicrobes” Candidate
Phyla Radiation (CPR) for the first time in human
stool samples. This “microscomics” approach is non-exhaustive
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promoters (katAp1 and katAp2). Here, we observed that KatA
was not required for acute virulence in another wild type P.
aeruginosa strain, PAO1, but that PAO1 exhibited higher
KatA expression than PA14 did. This was in a good agreement
with the observation that PAO1 was more resistant
than PA14 to H2O2 as well as to the antibiotic peptide, polymyxin
B (PMB), supposed to involve reactive oxygen species
(ROS) for its antibacterial activity. The higher KatA expression
in PAO1 than in PA14 was attributed to both katAp1
and katAp2 transcripts, as assessed by S1 nuclease mapping.
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to both katAp1 and katAp2 in a complementary manner
in PA14 and PAO1, by exploiting the promoter mutants
for each -10 box (p1m, p2m, and p1p2m). These results provide
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and Anr, which need to be further characterized for better
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Low-density lipoprotein (LDL) was recently reported to be an
opsonin, enhancing the phagocytosis of group A Streptococcus
(GAS) by human monocytic leukemia U937 cells due to the
binding of LDL to some GAS strains. We postulated that LDL
might also promote the opsonophagocytosis of Pseudomonas
aeruginosa by U937 cells since this bacterium interacts with
LDL. In this study, P. aeruginosa (CMCC10104), U937 cells,
and human LDL were used in phagocytosis assays to test our
hypothesis. Escherichia coli strain BL21, which does not interact
with LDL, was used as a negative control. Colony counting
and fluorescence microscopy were used to determine the
bacterial quantity in the opsonophagocytosis assays. After
incubation of U937 cells and P. aeruginosa with LDL (100
μg/ml) for 15 and 30 min, phagocytosis was observed to be
increased by 22.71% and 32.90%, respectively, compared to
that seen in the LDL-free group. However, LDL did not increase
the phagocytosis of E. coli by U937 cells. In addition,
we identified CD36 as a major opsonin receptor on U937 cells,
since an anti-CD36 monoclonal antibody, but not an anti-
CD4 monoclonal antibody, almost completely abolished the
opsonophagocytosis of P. aeruginosa by U937 cells.
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could disperse all types of biofilms, the thick flat biofilms
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Non-mammalian infection models have been developed over
the last two decades, which is a historic milestone to understand
the molecular basis of bacterial pathogenesis. They also
provide small-scale research platforms for identification of
virulence factors, screening for antibacterial hits, and evaluation
of antibacterial efficacy. The fruit fly, Drosophila melanogaster
is one of the model hosts for a variety of bacterial
pathogens, in that the innate immunity pathways and tissue
physiology are highly similar to those in mammals. We here
present a relatively simple protocol to assess the key aspects
of the polymicrobial interaction in vivo between the human
opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus
aureus, which is based on the systemic infection
by needle pricking at the dorsal thorax of the flies. After infection,
fly survival and bacteremia over time for both P.
aeruginosa and S. aureus within the infected flies can be monitored
as a measure of polymicrobial virulence potential.
The infection takes ~24 h including bacterial cultivation. Fly
survival and bacteremia are assessed using the infected flies
that are monitored up to ~60 h post-infection. These methods
can be used to identify presumable as well as unexpected phenotypes
during polymicrobial interaction between P. aeruginosa
and S. aureus mutants, regarding bacterial pathogenesis
and host immunity.
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Bacterial biofilms remain a persistent threat to human healthcare
due to their role in the development of antimicrobial
resistance. To combat multi-drug resistant pathogens, it is
crucial to enhance our understanding of not only the regulation
of biofilm formation, but also its contribution to bacterial
virulence. Iron acquisition lies at the crux of these two
subjects. In this review, we discuss the role of iron acquisition
in biofilm formation and how hosts impede this mechanism
to defend against pathogens. We also discuss recent findings
that suggest that biofilm formation can also have the reciprocal
effect, influencing siderophore production and iron
sequestration.
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In the bacterium Pseudomonas aeruginosa, the synthesis and
secretion of extracellular protease is a typical cooperative
behavior regulated by quorum sensing. However, this type
of cooperative behavior is easily exploited by other individuals
who do not synthesize public goods, which is known
as the “tragedy of the commons”. Here P. aeruginosa was inoculated
into casein media with different nitrogen salts added.
In casein broth, protease (a type of public good) is necessary
for bacterial growth. After 30 days of sequential transfer,
some groups propagated stably and avoided “tragedy of the
commons”. The evolved cooperators who continued to synthesize
protease were isolated from these stable groups. By
comparing the characteristics of quorum sensing in these
cooperators, an identical evolutionary pattern was found. A
variety of cooperative behaviors regulated by quorum sensing,
such as the synthesis and secretion of protease and signals,
were significantly reduced during the process of evolution.
Such reductions improved the efficiency of cooperation, helping
to prevent cheating. In addition, the production of pyocyanin,
which is regulated by the RhlIR system, increased
during the process of evolution, possibly due to its role in
stabilizing the cooperation. This study contributes towards
our understanding of the evolution of quorum sensing of P.
aeruginosa.
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Carbapenem-resistant Gram-negative bacteria (CR-GNB)
have emerged and disseminated worldwide, become a great
concern worldwide including Korea. The prevalence of fecal
carriage of imipenem-resistant Gram-negative bacteria (IRGNB)
in persons in Korea was investigated. Stool samples
were collected from 300 persons upon medical examination.
Samples were screened for IR-GNB by using MacConkey
agar with 2 μl/ml imipenem. Species were identified by 16S
rRNA gene sequence analysis, and antimicrobial susceptibility
was determined by the broth microdilution method.
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79 (26.3%) out of 300 healthy persons. Multilocus sequence
typing analysis showed very high diversity among IR P. aeruginosa,
S. maltophilia, and E. cloacae isolates, and pulsedfield
gel electrophoresis revealed five main pulsotypes of IR
P. mirabilis. As for the presence of metallo-β-lactamases
(MBLs), only one IMP-25-producing S. marcescens isolate
was identified. Although only one carbapenemase-producing
isolate was identified, the high colonization rates with IRGNB
isolates in this study is notable because carriers may
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Pseudomonas aeruginosa has been identified as an important
causative agent of airway infection, mainly in cystic fibrosis.
This disease is characterized by defective mucociliary clearance
induced in part by mucus hyper-production. Mucin is
a major component of airway mucus and is heavily O-glycosylated,
with a protein backbone. Airway infection is known
to be established with bacterial adhesion to mucin. However,
the genes involved in mucin degradation or utilization remain
elusive. In this study, we sought to provide a genetic basis of
P. aeruginosa airway growth by identifying those genes. First,
using RNASeq analyses, we compared genome-wide expression
profiles of PAO1, a prototype P. aeruginosa laboratory
strain, grown in M9-mucin (M9M) and M9-glucose (M9G)
media. Additionally, a PAO1 transposon (Tn) insertion mutants
library was screened for mutants defective in growth
in M9M medium. One mutant with a Tn insertion in the
xcpU gene (PA3100) was determined to exhibit faulty growth
in M9M medium. This gene contributes to the type II secretion
system, suggesting that P. aeruginosa uses this secretion
system to produce a number of proteins to break down and
assimilate the mucin molecule. Furthermore, we screened
the PAO1 genome for genes with protease activity. Of 13 mutants,
one with mutation in PA3247 gene exhibited defective
growth in M9M, suggesting that the PA3247-encoded protease
plays a role in mucin utilization. Further mechanistic
dissection of this particular process will reveal new drug targets,
the inhibition of which could control recalcitrant P. aeruginosa
infections.
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