When V. cholerae encounters nutritional stress, it activates (p)ppGpp-mediated stringent response. The genes relA and relV are involved in the production of (p)ppGpp, whereas the spoT gene encodes an ...enzyme that hydrolyzes it. Herein, we show that the bacterial capability to produce (p)ppGpp plays an essential role in glucose metabolism. The V. cholerae mutants defective in (p)ppGpp production (i.e. ΔrelAΔrelV and ΔrelAΔrelVΔspoT mutants) lost their viability because of uncontrolled production of organic acids, when grown with extra glucose. In contrast, the ΔrelAΔspoT mutant, a (p)ppGpp overproducer strain, exhibited better growth in the presence of the same glucose concentration. An RNA sequencing analysis demonstrated that transcriptions of genes consisting of an operon for acetoin biosynthesis were markedly elevated in N16961, a seventh pandemic O1 strain, but not in its (p)ppGpp0 mutant during glucose-stimulated growth. Transposon insertion in acetoin biosynthesis gene cluster resulted in glucose-induced loss of viability of the ΔrelAΔspoT mutant, further suggesting the crucial role of acetoin production in balanced growth under glucose-rich environments. Additional deletion of the aphA gene, encoding a negative regulator for acetoin production, failed to rescue the (p)ppGpp0 mutant from the defective glucose-mediated growth, suggesting that (p)ppGpp-mediated acetoin production occurs independent of the presence of AphA. Overall, our results reveal that (p)ppGpp, in addition to its well known role as a stringent response mediator, positively regulates acetoin production that contributes to the successful glucose metabolism and consequently the proliferation of V. cholerae cells under a glucose-rich environment, a condition that may mimic the human intestine.
Background: Bacteria respond to nutrient starvation by (p)ppGpp that activates the stringent response.
Results:V. cholerae mutants defective in (p)ppGpp production lost their viability during glucose-supplemented growth because of overproduction of organic acids.
Conclusion: (p)ppGpp regulates energy metabolism, contributing to the successful proliferation of V. cholerae under glucose-rich environments.
Significance: We report a previously unexplored role of (p)ppGpp in V. cholerae glucose metabolism.
Abstract
Broccoli extract (BE) has numerous beneficial effects on human health including anticancer activity. Quorum sensing (QS), mediated by self-produced autoinducer (AI) molecules, is a key ...process for the production of virulence determinants in pathogenic bacteria. BE suppressed AI-2 synthesis and AI-2-mediated bacterial motility in a dose-dependent manner in Escherichia coli O157:H7. In addition, expression of the ler gene that regulates AI-3 QS system was also diminished in response to treatment with BE. Furthermore, in an in vivo efficacy test using Caenorhabditis elegans as a host organism, C. elegans fed on E. coli O157:H7 in the presence of BE survived longer than those fed solely on the pathogenic bacteria. Quantitative real-time PCR analysis indicated that quercetin was the most active among the tested broccoli-derived compounds in downregulating virulence gene expression, while treatment with myricetin significantly suppressed the expression of the eae gene involved in type III secretion system. These data suggest that BE and its flavonoid constituents can inhibit expression of QS-associated genes, thereby downregulating the virulence attributes of E. coli O157:H7 both in vitro and in vivo. This study clearly elucidates BE's QS-inhibitory activity and suggests that BE has the potential to be developed as an anti-infective agent.
Pseudomonas aeruginosa frequently colonizes the lungs of chronic obstructive pulmonary disease (COPD) patients. Mucoid conversion is a hallmark of chronic P. aeruginosa infection, which is mediated ...by mucA gene mutations. The aim of this study is to identify predictive factors for mortality and the influence of mucA gene mutation in COPD patients with P. aeruginosa pneumonia.
This study assessed 75 COPD patients with P. aeruginosa pneumonia at two university hospitals. The clinical and laboratory data were collected, and the P. aeruginosa isolates analyzed for the presence of mucA gene mutations.
MucA gene mutation of P. aeruginosa was an independent predictor of mortality (odds ratio OR 10.43, 95% confidence interval CI: 1.53–70.90, p = 0.017). In addition, the APACHE II score and C-reactive protein/Albumin (CA) ratio were independent predictive factors for mortality (OR 1.25, 95% CI: 1.07–1.46, p = 0.004; and OR 1.06, 95% CI: 1.02–1.10, p = 0.003, respectively). The optimal cutoff value of CA ratio for the greatest sensitivity and specificity was calculated as 31.27 (sensitivity, 85.7%; specificity, 80.3%).
CA ratio and mucA gene mutation of P. aeruginosa could be used as predictors to identify poor prognosis in COPD patients with P. aeruginosa pneumonia.
•The presence of mucoid P. aeruginosa is associated with poor prognosis.•Mucoid conversion of P. aeruginosa is mediated by MucA gene mutation.•MucA gene mutation of P. aeruginosa is an independent predictor of mortality.
Pseudomonas aeruginosa is an opportunistic pathogen, known to develop robust biofilms. Its biofilm development increases when antibiotics are presented at subminimal inhibitory concentrations (MICs) ...for reasons that remain unclear. In order to identify genes that affect biofilm development under such a sublethal antibiotic stress condition, we screened a transposon (Tn) mutant library of PAO1, a prototype P. aeruginosa strain. Among ∼5000 mutants, a fiuA gene mutant was verified to form very defective biofilms in the presence of sub-MIC carbenicillin. The fiuA gene encodes ferrichrome receptor A, involved in the iron acquisition process. Of note, biofilm formation was not decreased in the ΔpchΔpvd mutant defective in the production of pyochelin and pyoverdine, two well-characterized P. aeruginosa siderophore molecules. Moreover, ΔfiuA, a non-polar fiuA deletion mutant, produced a significantly decreased level of elastase, a major virulence determinant. Mouse airway infection experiments revealed that the mutant expressed significantly less pathogenicity. Our results suggest that the fiuA gene has pleiotropic functions that affect P. aeruginosa biofilm development and virulence. The targeting of FiuA could enable the attenuation of P. aeruginosa virulence and may be suitable for the development of a drug that specifically controls the virulence of this important pathogen.
Antibiotics stimulate biofilm formation in P. aeruginosa for reasons to be further explored. A mutant of ferrichrome receptor A was defective in antibiotic-inducible biofilm formation and exhibited concomitant virulence attenuation.
Summary
Caenorhabditis elegans, originally isolated from soil, is a nematode used in various fields of biological research including host–microbe interaction. While bacterial pathogens responsible ...for human infections have been actively studied in C. elegans, very few bacterial species that provide beneficial effects on C. elegans have been reported. Here, we tested several bacterial soil isolates and then characterized the effects of Lysinibacillus sphaericus on C. elegans growth‐related phenotypes. Worms fed with L. sphaericus lived significantly longer than those growing with typical Escherichia coli OP50. Early‐ and juvenile‐stage growth was also highly stimulated by L. sphaericus; body size at 28 h post‐hatching was > 2 times larger than OP50‐fed worms and L. sphaericus‐fed worms moved through the larval stage development more rapidly than control worms. In addition, significantly elevated fertilization was observed in worms fed with L. sphaericus (∼ 8 h faster than the control group). Furthermore, growth with L. sphaericus resulted in the production of larger numbers of progeny than the control growth with OP50. Worms grown with L. sphaericus were highly resistant to oxidative, osmotic and infection stresses. Together, our results reveal a novel mode of growth that involves healthy ageing of nematodes.
Transcription factors are the primary regulators of gene expression and recognize specific DNA sequences under diverse physiological conditions. Although they are vital for many important cellular ...processes, it remains unclear when and how transcription factors and DNA interact. The antitoxin from a toxin–antitoxin system is an example of negative transcriptional autoregulation: during expression of the cognate toxin it is suppressed through binding to a specific DNA sequence. In the present study, the antitoxin HigA2 from
Mycobacterium tuberculosis
M37Rv was structurally examined. The crystal structure of
M. tuberculosis
HigA2 comprises three sections: an N-terminal autocleavage region, an α-helix bundle which contains an HTH motif, and a C-terminal β-lid. The N-terminal region is responsible for toxin binding, but was shown to cleave spontaneously in its absence. The HTH motif performs a key role in DNA binding, with the C-terminal β-lid influencing the interaction by mediating the distance between the motifs. However,
M. tuberculosis
HigA2 exhibits a unique coordination of the HTH motif and no DNA-binding activity is detected. Three crystal structures of
M. tuberculosis
HigA2 show a flexible alignment of the HTH motif, which implies that the motif undergoes structural rearrangement to interact with DNA. This study reveals the molecular mechanisms of how transcription factors interact with partner proteins or DNA.
The human microbiome contains genetic information that regulates metabolic processes in response to host health and disease. While acidic vaginal pH is maintained in normal conditions, the pH level ...increases in infectious vaginitis. We propose that this change in the vaginal environment triggers the biosynthesis of anti-vaginitis metabolites. Gene expression levels of Chryseobacterium gleum, a vaginal symbiotic bacterium, were found to be affected by pH changes. The distinctive difference in the metabolic profiles between two C. gleum cultures incubated under acidic and neutral pH conditions was suggested to be an anti-vaginitis molecule, which was identified as phenylacetic acid (PAA) by spectroscopic data analysis. The antimicrobial activity of PAA was evaluated in vitro, showing greater toxicity toward Gardnerella vaginalis and Candida albicans, two major vaginal pathogens, relative to commensal Lactobacillus spp. The activation of myeloperoxidase, prostaglandin E
, and nuclear factor-κB, and the expression of cyclooxygenase-2 were reduced by an intravaginal administration of PAA in the vaginitis mouse model. In addition, PAA displayed the downregulation of mast cell activation. Therefore, PAA was suggested to be a messenger molecule that mediates interactions between the human microbiome and vaginal health.
As a facultative anaerobe, Vibrio cholerae can grow by anaerobic respiration. Production of cholera toxin (CT), a major virulence factor of V. cholerae, is highly promoted during anaerobic growth ...using trimethylamine N-oxide (TMAO) as an alternative electron acceptor. Here, we investigated the molecular mechanisms of TMAO-stimulated CT production and uncovered the crucial involvement of stringent response in this process. V. cholerae 7th pandemic strain N16961 produced a significantly elevated level of ppGpp, the bacterial stringent response alarmone, during anaerobic TMAO respiration. Bacterial viability was impaired, and DNA replication was also affected under the same growth condition, further suggesting that stringent response is induced. A ΔrelA ΔspoT ppGpp overproducer strain produced an enhanced level of CT, whereas anaerobic growth via TMAO respiration was severely inhibited. In contrast, a ppGpp-null strain (ΔrelA ΔspoT ΔrelV) grew substantially better, but produced no CT, suggesting that CT production and bacterial growth are inversely regulated in response to ppGpp accumulation. Bacterial capability to produce CT was completely lost when the dksA gene, which encodes a protein that works cooperatively with ppGpp, was deleted. In the ΔdksA mutant, stringent response growth inhibition was alleviated, further supporting the inverse regulation of CT production and anaerobic growth. In vivo virulence of ΔrelA ΔspoT ΔrelV or ΔdksA mutants was significantly attenuated. The ΔrelA ΔspoT mutant maintained virulence when infected with exogenous TMAO despite its defective growth. Together, our results reveal that stringent response is activated under TMAO-stimulated anaerobic growth, and it regulates CT production in a growth-dependent manner in V. cholerae.
Cholera toxin (CT) production is induced during anaerobic respiration with trimethylamine N-oxide (TMAO) in Vibrio cholerae.
A bacterial stringent response to nutrient starvation was activated during anaerobic TMAO respiration and influenced CT production.
CT production during anaerobic TMAO respiration is mediated by stringent response in V. cholerae.
A mechanism of TMAO-stimulated CT production is uncovered.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation to human society through its high virulence, infectivity, and genomic mutations, which reduced the efficacy of ...vaccines. Here, we report the development of aptamers that effectively interfere with SARS-CoV-2 infection by targeting its spike protein, which plays a pivotal role in host cell entry of the virus through interaction with the viral receptor angiotensin-converting enzyme 2 (ACE2). To develop highly effective aptamers and to understand their mechanism in inhibiting viral infection, we determined the three-dimensional (3D) structures of aptamer/receptor-binding domain (RBD) complexes using cryogenic electron microscopy (cryo-EM). Moreover, we developed bivalent aptamers targeting two distinct regions of the RBD in the spike protein that directly interact with ACE2. One aptamer interferes with the binding of ACE2 by blocking the ACE2-binding site in RBD, and the other aptamer allosterically inhibits ACE2 by binding to a distinct face of RBD. Using the 3D structures of aptamer-RBD complexes, we minimized and optimized these aptamers. By combining the optimized aptamers, we developed a bivalent aptamer that showed a stronger inhibitory effect on virus infection than the component aptamers. This study confirms that the structure-based aptamer-design approach has a high potential in developing antiviral drugs against SARS-CoV-2 and other viruses.
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