The production of recombinant proteins in Escherichia coli is often challenged by cytoplasmic expression due to proteolytic degradation and inclusion body formation. Extracellular expression can overcome these problems by simplifying downstream processing and improving protein yields. This study aims to compare the efficiency of two Bacillus subtilis chitosanase signal peptides in mediating extracellular secretion in E. coli. We identified a naturally occurring mutant signal peptide (mCsn2-SP) from B. subtilis CH2 chitosanase (CH2CSN), which is characterized by a deletion of six amino acids in the N-region relative to the signal peptide (Csn1-SP) from B. subtilis CH1 chitosanase (CH1CSN). The CH1CSN and CH2CSN genes were cloned into the pET-11a vector and protein secretion was evaluated in E. coli BL21(DE3) host cells. Expression was induced with 0.1 mM and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30 °C for one and three days. CH2CSN showed higher secretion levels compared to CH1CSN under all experimental conditions, especially with 0.1 mM IPTG induction for 3 days, which resulted in a 2.37-fold increase in secretion. Furthermore, it was demonstrated that mCsn2-SP is capable of secreting human Cu,Zn-superoxide dismutase (hSOD) in E. coli BL21(DE3) and successfully translocating it to the periplasmic region.
This study represents the inaugural investigation into the utilisation of a naturally modified signal peptide, thereby corroborating the assertion that signal peptide deletion variants can influence protein secretion efficiency.
Furthermore, the findings substantiate the proposition that such variants can serve as a viable alternative for the secretion of heterologous proteins in E.
coli.
Over the past two decades, as the importance of gut microbiota to human health has become widely known, attempts have been made to treat diseases by correcting dysbiosis of gut microbiota through fecal microbiota transplantation (FMT). Apart from current knowledge of gut microbiota, FMT to treat disease has a long history, from the treatment of food poisoning in the fourth century to the treatment of Clostridioides difficile infections in the twentieth century. In 2013, FMT was recognized as a standard treatment for recurrent C. difficile because it consistently showed high efficacy. Though recurrent C. difficile is the only disease internationally recognized for FMT efficacy, FMT has been tested for other diseases and shown some promising preliminary results. Different FMT methods have been developed using various formulations and administration routes.
Despite advances in FMT, some issues remain to be resolved, such as donor screening, manufacturing protocols, and unknown components in the fecal microbiota. In this review, we discuss the mechanisms, clinical indications, methods, and challenges of current FMT. We also discuss the development of alternative therapies to overcome the challenges of FMT.
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Koketso Desiree Mazwi, Kgaugelo Edward Lekota, Barbara Akofo Glover, Francis Babaman Kolo, Ayesha Hassim, Jenny Rossouw, Annelize Jonker, Justnya Maria Wojno, Giuseppe Profiti, Pier Luigi Martelli, Rita Casadio, Katiuscia Zilli, Anna Janowicz, Francesca Marotta, Giuliano Garofolo, Henriette van Heerden
J. Microbiol. 2024;62(9):759-773. Published online July 22, 2024
Brucellosis is an economically important zoonotic disease affecting humans, livestock, and wildlife health globally and especially in Africa. Brucella abortus and B. melitensis have been isolated from human, livestock (cattle and goat), and wildlife (sable) in South Africa (SA) but with little knowledge of the population genomic structure of this pathogen in SA. As whole genome sequencing can assist to differentiate and trace the origin of outbreaks of Brucella spp.
strains, the whole genomes of retrospective isolates (n = 19) from previous studies were sequenced. Sequences were analysed using average nucleotide identity (ANI), pangenomics, and whole genome single nucleotide polymorphism (wgSNP) to trace the geographical origin of cases of brucellosis circulating in human, cattle, goats, and sable from different provinces in SA. Pangenomics analysis of B. melitensis (n = 69) and B. abortus (n = 56) was conducted with 19 strains that included B. abortus from cattle (n = 3) and B. melitensis from a human (n = 1), cattle (n = 1), goat (n = 1), Rev1 vaccine strain (n = 1), and sable (n = 12).
Pangenomics analysis of B. melitensis genomes, highlighted shared genes, that include 10 hypothetical proteins and genes that encodes for acetyl-coenzyme A synthetase (acs), and acylamidase (aam) amongst the sable genomes. The wgSNP analysis confirmed the B. melitensis isolated from human was more closely related to the goat from the Western Cape Province from the same outbreak than the B.
melitensis cattle sample from different cases in the Gauteng Province. The B.
melitensis sable strains could be distinguished from the African lineage, constituting their own African sub-clade. The sequenced B. abortus strains clustered in the C2 lineage that is closely related to the isolates from Mozambique and Zimbabwe. This study identified genetically diverse Brucella spp.
among various hosts in SA. This study expands the limited known knowledge regarding the presence of B. melitensis in livestock and humans in SA, further building a foundation for future research on the distribution of the Brucella spp. worldwide and its evolutionary background.
Most microorganisms resist pure cultivation under conventional laboratory conditions. One of the primary issues for this un-culturability is the absence of biologically produced growth-promoting factors in traditionally defined growth media. However, whether cultivating microbes by providing spent culture supernatant of pivotal microbes in the growth medium can be an effective approach to overcome this limitation is still an under-explored area of research. Here, we used the spent culture medium (SCM) method to isolate previously uncultivated marine bacteria and compared the efficiency of this method with the traditional cultivation (TC) method. In the SCM method, Ca. Bathyarchaeia-enriched supernatant (10%) was used along with recalcitrant organic substrates such as lignin, humic acid, and organic carbon mixture. Ca. Bathyarchaeia, a ubiquitous class of archaea, have the capacity to produce metabolites, making their spent culture supernatant a key source to recover new bacterial stains. Both cultivation methods resulted in the recovery of bacterial species from the phyla Pseudomonadota, Bacteroidota, Actinomycetota, and Bacillota. However, our SCM approach also led to the recovery of species from rarely cultivated groups, such as Planctomycetota, Deinococcota, and Balneolota. In terms of the isolation of new taxa, the SCM method resulted in the cultivation of 80 potential new strains, including one at the family, 16 at the genus, and 63 at the species level, with a novelty ratio of ~ 35% (80/219). In contrast, the TC method allowed the isolation of ~ 10% (19/171) novel strains at species level only. These findings suggest that the SCM approach improved the cultivation of novel and diverse bacteria.
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Two bacterial strains (XCT-34T and XCT-53) isolated from sediment samples of an artificial freshwater reservoir were analyzed using a polyphasic approach. The two isolates are aerobic, Gram-stain-negative, oxidase-negative, catalase-positive, motile with polar flagella, rod-shaped, and approximately 1.4-3.4 × 0.4-0.9 μm in size. Phylogenetic analyses based on 16S rRNA gene and whole-genome sequences showed that the two strains formed a distinct branch within the evolutionary radiation of the genus Pannonibacter, closest to Pannonibacter carbonis Q4.6T (KCTC 52466). Furthermore, lower than threshold average nucleotide identity values (ANI, 85.7-86.4%) and digital DNA-DNA hybridization values (dDDH, 22.3-30.5%) of the two strains compared to the nearest type strains also confirmed that they represented a novel species.
Genomic analyses, including annotation of the KEGG pathways, prediction of the secondary metabolism biosynthetic gene clusters and PHI phenotypes, supported functional inference and differentiation of the strains from the closely related taxa. Results of chemotaxonomic and physiological studies revealed that their distinct phenotypic characteristics distinguished them from existing Pannonibacter species. Thus, the two strains are considered to represent a novel species of Pannonibacter, for which the name of Pannonibacter tanglangensis sp.
nov. is proposed, with XCT-34T (= KCTC 82332T = GDMCC 1.1947T) as the respective type strain.
Extracellular vesicles (EVs), of diverse origin and content, are membranous structures secreted by a broad range of cell types. Recent advances in molecular biology have highlighted the pivotal role of EVs in mediating intercellular communication, facilitated by their ability to transport a diverse range of biomolecules, including proteins, lipids, DNA, RNA and metabolites. A striking feature of EVs is their ability to exert dual effects during viral infections, involving both proviral and antiviral effects. This review explores the dual roles of EVs, particularly in the context of pandemic viruses such as HIV-1 and SARS-CoV-2. On the one hand, EVs can enhance viral replication and exacerbate pathogenesis by transferring viral components to susceptible cells. On the other hand, they have intrinsic antiviral properties, including activation of immune responses and direct inhibition of viral infection. By exploring these contrasting functions, our review emphasizes the complexity of EV-mediated interactions in viral pathogenesis and highlights their potential as targets for therapeutic intervention. The insights obtained from investigating EVs in the context of HIV-1 and SARS-CoV-2 provide a deeper understanding of viral mechanisms and pathologies, and offer a new perspective on managing and mitigating the impact of these global health challenges.
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This study aimed to develop synthetic Claudin18.2 (CLDN18.2) chimeric antigen receptor (CAR)-T (CAR-T) cells as a treatment for advanced gastric cancer using lentiviral vector genetic engineering technology that targets the CLDN18.2 antigen and simultaneously overcomes the immunosuppressive environment caused by programmed cell death protein 1 (PD-1). Synthetic CAR T cells are a promising approach in cancer immunotherapy but face many challenges in solid tumors. One of the major problems is immunosuppression caused by PD-1. CLDN18.2, a gastric-specific membrane protein, is considered a potential therapeutic target for gastric and other cancers. In our study, CLDN18.2 CAR was a second-generation CAR with inducible T-cell costimulatory (CD278), and CLDN18.2-PD1/CD28 CAR was a third-generation CAR, wherein the synthetic PD1/CD28 chimeric-switch receptor (CSR) was added to the second-generation CAR. In vitro, we detected the secretion levels of different cytokines and the killing ability of CAR-T cells. We found that the secretion of cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) secreted by three types of CAR-T cells was increased, and the killing ability against CLDN18.2-positive GC cells was enhanced. In vivo, we established a xenograft GC model and observed the antitumor effects and off-target toxicity of CAR-T cells. These results support that synthetic anti-CLDN18.2 CAR-T cells have antitumor effect and anti-CLDN18.2-PD1/CD28 CAR could provide a promising design strategy to improve the efficacy of CAR-T cells in advanced gastric cancer.
Nitrate (
NO3
−) is highly water-soluble and considered to be the main nitrogen pollutants leached from agricultural soils. Its
presence in aquatic ecosystems is reported to cause various environmental and public health problems. Bioreactors containing
microbes capable of transforming NO3
− have been proposed as a means to remediate contaminated waters. Woodchip bioreactors
(WBRs) are continuous flow, reactor systems located below or above ground. Below ground systems are comprised
of a trench filled with woodchips, or other support matrices. The nitrate present in agricultural drainage wastewater passing
through the bioreactor is converted to harmless dinitrogen gas (
N2) via the action of several bacteria species. The WBR has
been suggested as one of the most cost-effective NO3
−-removing strategy among several edge-of-field practices, and has been
shown to successfully remove NO3
− in several field studies. NO3
− removal in the WBR primarily occurs via the activity of
denitrifying microorganisms via enzymatic reactions sequentially reducing NO3
− to N2.
While previous woodchip bioreactor
studies have focused extensively on its engineering and hydrological aspects, relatively fewer studies have dealt with the
microorganisms playing key roles in the technology. This review discusses NO3
− pollution cases originating from intensive
farming practices and N-cycling microbial metabolisms which is one biological solution to remove NO3
− from agricultural
wastewater. Moreover, here we review the current knowledge on the physicochemical and operational factors affecting
microbial metabolisms resulting in removal of NO3
− in WBR, and perspectives to enhance WBR performance in the future.
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In the post-genomic era, phylogenomics is a powerful and routinely-used tool to discover evolutionary relationships between
microorganisms. Inferring phylogenomic trees by concatenating core gene sequences into a supermatrix is the standard
method . The previously released up-to-date bacterial core gene (UBCG) tool provides a pipeline to infer phylogenomic trees
using single-copy core genes for the Bacteria domain. In this study, we established up-to-date archaeal core gene (UACG),
comprising 128 genes suitable for inferring archaeal phylogenomic trees. To test the gene set, we selected the Haloarcula
genus and scrutinized its phylogeny. The phylogeny inferred using the UACG tool was consistent with the orthoANIu
dendrogram, whereas the 16S rRNA gene phylogeny showed high intragenomic heterogeneity resulting in phylogenetic
discrepancies. The software tool using the UACG set is available at https:// www. ezbio cloud. net/ tools/ uacg.
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The Fusarium graminearum species complex (FGSC) can produce various mycotoxins and is a major concern for food
quantity and quality worldwide. In this study, we determined the effects of water activity (
aw), temperature, incubation time
and their interactions on mycotoxin accumulation and the expression levels of biosynthetic genes in FGSC strains from
maize samples in China. The highest deoxynivalenol (DON), 3-acetyldeoxynivalenol(3ADON) and 15-acetyldeoxynivalenol
(15ADON) levels of the F. boothii and F. graminearum strains were observed at 0.98 aw/
30 °C or 0.99 aw/
25 °C. F. asiaticum
and F. meridionale reached maximum nivalenol (NIV) and 4-acetylnivalenol (4ANIV) contents at 0.99 aw
and 30 °C. With
the extension of the incubation time, the concentrations of DON and NIV gradually increased, while those of their derivatives
decreased. F. boothii, F. meridionale and one F. asiaticum strain had the highest zearalenone (ZEN) values at 0.95 aw
and 25 °C, while the optimum conditions for the other F. asiaticum strain and F. graminearum were 0.99 aw
and 30 °C. Four
genes associated with trichothecene and zearalenone synthesis were significantly induced under higher water stress in the
early stage of production. The results indicated independence of mycotoxin production and gene expression, as maximum
amounts of these toxic metabolites were observed at higher aw
in most cases. This study provides useful information for the
monitoring and prevention of such toxins entering the maize production chain.
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Microbial communities played a vital role in maintaining homeostasis of ocular surface. However, no studies explored the
myopia-associated conjunctiva microbiota changes until now. In this study, conjunctival sac swab specimens were collected
from 12 eyes of low myopia (LM), and 14 eyes of high myopia (HM) patients. The V3–V4 region of the 16S rRNA gene was
amplified and then sequenced. Statistical analysis was performed to investigate differences in the taxonomy and diversity
between two groups. Compared to LM, higher Ocular Surface Disease Index (OSDI) scores were observed in HM group. The
Shannon index of the HM was lower than that of the LM group (P = 0.017). Principle coordinate analysis and Partial Least
Squares Discrimination Analysis showed distinct microbiome composition between two groups. At the phylum level, there
were higher relative abundances of Proteobacteria (68.27% vs 38.51%) and lower abundances of Actinobacteria (3.71% vs
9.19%) in HM, compared to LM group (P = 0.031, 0.010, respectively). At the genus level, the abundances of Acinetobacter
in HM (18.16%) were significantly higher than the LM (6.52%) group (P = 0.011). Actinobacteria levels were negatively
correlated with the myopic spherical equivalent and OSDI scores. Moreover, positive correlations were found between
Proteobacteria levels and OSDI scores, Acinetobacter levels were positively correlated with myopic spherical equivalent
and OSDI scores. In conclusion, HM Patients have bacterial microbiota imbalance in the conjunctival sac, compared with
LM patients. Proteobacteria, Actinobacteria, Acinetobacter may play roles in the HM associated ocular surface irritation.
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Vaginal microbiome composition was demonstrated to be associated with cervical disease. The colonization characteristics
of vaginal microbes and their association with the different cervical disease status, especially cervical cancer (CC), are
rarely investigated. In this cross-sectional study, we characterized the vaginal microbiome of women with different status of
cervical diseases, including 22 NV + (normal tissue with HPV infection), low-grade squamous intraepithelial lesion (LSIL,
n = 45), high-grade squamous intraepithelial lesion (HSIL, n = 36) and CC (n = 27) using bacterial 16S DNA sequencing.
Thirty HPV-negative women with normal tissue were used as the control group. We found that higher diversity of microbiome
with gradual depletion of Lactobacillus, especially L. crispatus, was associated with the severity of cervical disease.
High-risk HPV16 infection was associated with higher microbiome diversity and depletion of Lactobacillus in high-grade
cervical diseases (i.e. HSIL and CC). The CC group was characterized by higher levels of Fannyhessea vaginae, Prevotella,
Bacteroides, Finegoldia, Vibrio, Veillonella, Peptostreptococcus, and Dialister. Co-occurrence network analyses showed that
negative correlations were exclusively observed between Lactobacillus and other bacteria, and almost all non-Lactobacillus
bacteria were positively correlated with each other. In particular, the most diverse and complex co-occurrence network of
vaginal bacteria, as well as a complete loss of L. crispatus, was observed in women with CC. Logistic regression model
identified HPV16 and Lactobacillus as significant risk and protective factors for CC, respectively. These results suggest that
specific Lactobacillus species (e.g. L. crispatus and L. iners) can be used as important markers to target prevention measures
prioritizing HPV16-infected women and other hrHPV-infected women for test, vaccination and treat initiatives.
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The morphological switch from the yeast to hyphal form is a key virulence attribute of the opportunistic fungal pathogen,
Candida albicans. Our recent report showed that deletion of the newly identified apoptotic factor, CaNma111 or CaYbh3,
leads to hyperfilamentation and increased virulence in a mouse infection model. CaNma111 and CaYbh3 are homologs of the
pro-apoptotic protease, HtrA2/Omi, and BH3-only protein, respectively. In this study, we examined the effects of CaNMA111
and CaYBH3 deletion mutations on the expression levels of the hypha-specific transcr!ption factors, Cph1 (a hyphal activator),
Nrg1 (a hyphal repressor), and Tup1 (a hyphal repressor). The protein levels of Nrg1 were decreased in Caybh3/Caybh3 cells
while those of Tup1 were decreased in both Canma111/Canma111 and Caybh3/Caybh3 cells. These effects on Nrg1 and
Tup1 proteins were retained during serum-induced filamentation and appear to explain the hyperfilamentation phenotypes
of the CaNMA111 and CaYBH3 deletion mutants. Treatment with the apoptosis-inducing dose of farnesol decreased the
Nrg1 protein levels in the wild-type strain and more evidently in Canma111/Canma111 and Caybh3/Caybh3 mutant strains.
Together, our results suggest that CaNma111 and CaYbh3 are key regulators of Nrg1 and Tup1 protein levels in C. albicans.
Bacterial cells are covered with various glycopolymers such as peptidoglycan (PG), lipopolysaccharides (LPS), teichoic
acids, and capsules. Among these glycopolymers, PG assembly is the target of some of our most effective antibiotics, consistent
with its essentiality and uniqueness to bacterial cells. Biosynthesis of other surface glycopolymers have also been
acknowledged as potential targets for developing therapies to control bacterial infections, because of their importance for
bacterial survival in the host environment. Moreover, biosynthesis of most surface glycopolymers are closely related to PG
assembly because the same lipid carrier is shared for glycopolymer syntheses. In this review, I provide an overview of PG
assembly and antibiotics that target this pathway. Then, I discuss the implications of a common lipid carrier being used for
assembly of PG and other surface glycopolymers in antibiotic development.
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RNase E is an essential enzyme in Escherichia coli. The cleavage site of this single-stranded specific endoribonuclease is
well-characterized in many RNA substrates. Here, we report that the upregulation of RNase E cleavage activity by a mutation
that affects either RNA binding (Q36R) or enzyme multimerization (E429G) was accompanied by relaxed cleavage specificity.
Both mutations led to enhanced RNase E cleavage in RNA I, an antisense RNA of ColE1-type plasmid replication,
at a major site and other cryptic sites. Expression of a truncated RNA I with a major RNase E cleavage site deletion at the
5′-end (RNA I-
5) resulted in an approximately twofold increase in the steady-state levels of RNA I-
5 and the copy number
of ColE1-type plasmid in E. coli cells expressing wild-type or variant RNase E compared to those expressing RNA I. These results indicate that RNA I-
5 does not efficiently function as an antisense RNA despite having a triphosphate group at the
5′-end, which protects the RNA from ribonuclease attack. Our study suggests that increased cleavage rates of RNase E lead
to relaxed cleavage specificity on RNA I and the inability of the cleavage product of RNA I as an antisense regulator in vivo
does not stem from its instability by having 5′-monophosphorylated end.
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