A review and new perspective on oral bacteriophages: manifestations in the ecology of oral diseases.

Objective: To explore the manifestations of bacteriophages in different oral disease ecologies, including periodontal diseases, dental caries, endodontic infections, and oral cancer, as well as to propel phage therapy for safer and more effective clinical application in the field of dentistry. Methods: In this literature review, we outlined interactions between bacteriophages, bacteria and even oral cells in the oral ecosystem, especially in disease states. We also analyzed the current status and future prospects of phage therapy in the perspective of different oral diseases. Results: Various oral bacteriophages targeting at periodontal pathogens as Porphyromonas gingivalis, Fusobacterium nucleatum, Treponema denticola and Aggregatibacter actinomycetemcomitans, cariogenic pathogen Streptococcus mutans, endodontic pathogen Enterococcus faecalis were predicted or isolated, providing promising options for phage therapy. In the realm of oral cancer, aside from displaying tumor antigens or participating in tumor-targeted therapies, phage-like particle vaccines demonstrated the potential to prevent oral infections caused by human papillomaviruses (HPVs) associated with head-and-neck cancers. Conclusion: Due to their intricate interactions with bacteria and oral cells, bacteriophages are closely linked to the progression and regression of diverse oral diseases. And there is an urgent need for research to explore additional possibilities of bacteriophages in the management of oral diseases.

Inhibition of Streptococcus mutans growth and biofilm formation through protein acetylation.

Numerous cellular processes are regulated in response to the metabolic state of the cell, and one such regulatory mechanism involves lysine acetylation. Lysine acetylation has been proven to play an important role in the virulence of Streptococcus mutans, a major cariogenic bacterial species. S. mutans' glucosyltransferases (Gtfs) are responsible for synthesizing extracellular polysaccharides (EPS) and contributing to biofilm formation. One of the most common nonsteroidal anti-inflammatory drugs is acetylsalicylic acid (ASA), which can acetylate proteins through a nonenzymatic transacetylation reaction. Herein, we investigated the inhibitory effects of ASA on S. mutans. ASA treatment was observed to impede the growth of S. mutans, leading to a reduction in the production of water-insoluble EPS and the formation of biofilm. Moreover, ASA decreased the enzyme activity of Gtfs while increasing the protein acetylation level. The in vivo anticaries efficacy of ASA has further been proved using the rat caries model. In conclusion, ASA as an acetylation agent attenuated the cariogenic virulence of S. mutans, suggesting the potential value of protein acetylation on antimicrobial and anti-biofilm applications to S. mutans.

Discovery and Mechanistic Study of Novel Mycobacterium tuberculosis ClpP1P2 Inhibitors.

Caseinolytic protease P (ClpP) responsible for the proteolysis of damaged or misfolded proteins plays a critical role in proteome homeostasis. MtbClpP1P2, a ClpP enzyme complex, is required for survival in Mycobacterium tuberculosis, and it is therefore considered as a promising target for the development of antituberculosis drugs. Here, we discovered that cediranib and some of its derivatives are potent MtbClpP1P2 inhibitors and suppress M. tuberculosis growth. Protein pull-down and loss-of-function assays validated the in situ targeting of MtbClpP1P2 by cediranib and its active derivatives. Structural and mutational studies revealed that cediranib binds to MtbClpP1P2 by binding to an allosteric pocket at the equatorial handle domain of the MtbClpP1 subunit, which represents a unique binding mode compared to other known ClpP modulators. These findings provide us insights for rational drug design of antituberculosis therapies and implications for our understanding of the biological activity of MtbClpP1P2.

Discovery of novel reversible inhibitor of DprE1 based on benzomorpholine for the treatment of tuberculosis.

About a quarter of the world's population is infected with Mycobacterium tuberculosis, equivalent to about two billion people. With the emergence of multidrug-resistant tuberculosis, those existing anti-tuberculosis drugs no longer meet the demand for cure anymore; there is an urgent need for the development of new anti-tuberculosis drugs. Decaprenylphosphoryl-ß-D-ribose 2´-epimerase (DprE1) has been proven to be a potential antimycobacterial target, and several inhibitors have entered clinical trial. Herein, we designed and synthesized a series of compounds based on the indole and benzomorpholine by using the strategy of scaffold hopping. The preferred compound B18 showed strong antimycobacterial activity in H37Rv and drug-resistant clinical isolates. In addition, compound B18 did not exhibit antimycobacterial efficacy against other species of strains. Subsequently, the target of B18 was identified as DprE1 by analyzing spontaneous compound-resistant mutation data, and a docking study was performed to illustrate the binding mode between B18 and DprE1. In general, compound B18 is compatible to current DprE1 inhibitors, even higher phosphodiesterase 6C selectivity and plasma protein binding rate, which represent a new type of effective reversible DprE1 inhibitor. IMPORTANCE Drug therapy remains the cornerstone of tuberculosis (TB) treatment, yet first-line anti-tuberculosis drugs are associated with significant adverse effects that can compromise patient outcomes. Moreover, prolonged and widespread use has led to an alarming rise in drug-resistant strains of Mycobacterium tuberculosis, including multidrug-resistant [MDR-tuberculosis (TB)] and extensively drug-resistant (XDR-TB) forms. Urgent action is needed to develop novel anti-tuberculosis agents capable of overcoming these challenges. We report that compound B18, a decaprenylphosphoryl-ß-D-ribose 2´-epimerase inhibitor with a benzomorpholine backbone, exhibits potent activity against not only the non-pathogenic strain H37Ra, but also the pathogenic strain H37Rv and clinical MDR and XDR strains. Preliminary druggability studies indicate that B18 possesses high safety and acceptable pharmacokinetic properties, rendering it a promising candidate for further development as a novel anti-tuberculosis agent.

Membrane vesicles derived from Listeria monocytogenes might be a potential antigen delivery vector.

Membrane vesicles (MVs) derived from Listeria monocytogenes (LM) have a natural nanoscale size and contain a variety of bacterial components. We speculated that LM MVs may be a novel delivery vector, but it is necessary to evaluate the safety and immunogenicity of LM MVs in vivo. Here, we isolated LM MVs and tested their safety and immunogenicity both in vitro and in vivo. The results showed that LM MVs stimulated RAW264.7 cells and DC2.4 cells to secrete the inflammatory cytokines IL-1ß, TNF-α, IL-6 and IL-10. Intraperitoneal injection of LM MVs at 80 µg per C57BL/6 mouse did not cause lethal effects or irreversible pathological changes in major organs, indicating that LM MVs were safe. Intraperitoneal immunization of C57BL/6 mice twice with LM MVs mainly induced a high level of LM MV-specific IgG antibodies. In addition, we subcutaneously injected C57BL/6 mice with a mixture of ovalbumin and LM MVs and found that LM MVs exhibited a humoral immune adjuvant effect equal to that of the same amount of alum. The results of this study indicated that LM MVs have good safety and effective immunogenicity and may act as humoral immune adjuvants. Therefore, LM MVs are a potential new choice for antigen and drug delivery vectors.

Regulatory mechanisms of exopolysaccharide synthesis and biofilm formation in Streptococcus mutans.

Background: Dental caries is a chronic, multifactorial and biofilm-mediated oral bacterial infection affecting almost every age group and every geographical region. Streptococcus mutans is considered an important pathogen responsible for the initiation and development of dental caries. It produces exopolysaccharides in situ to promote the colonization of cariogenic bacteria and coordinate dental biofilm development. Objective: The understanding of the regulatory mechanism of S. mutans biofilm formation can provide a theoretical basis for the prevention and treatment of caries. Design: At present, an increasing number of studies have identified many regulatory systems in S. mutans that regulate biofilm formation, including second messengers (e.g. c-di-AMP, Ap4A), transcription factors (e.g. EpsR, RcrR, StsR, AhrC, FruR), two-component systems (e.g. CovR, VicR), small RNA (including sRNA0426, srn92532, and srn133489), acetylation modifications (e.g. ActG), CRISPR-associated proteins (e.g. Cas3), PTS systems (e.g. EIIAB), quorum-sensing signaling system (e.g. LuxS), enzymes (including Dex, YidC, CopZ, EzrA, lmrB, SprV, RecA, PdxR, MurI) and small-molecule metabolites. Results: This review summarizes the recent progress in the molecular regulatory mechanisms of exopolysaccharides synthesis and biofilm formation in S. mutans.

Crosstalk between microbial biofilms in the gastrointestinal tract and chronic mucosa diseases.

The gastrointestinal (GI) tract is the largest reservoir of microbiota in the human body; however, it is still challenging to estimate the distribution and life patterns of microbes. Biofilm, as the predominant form in the microbial ecosystem, serves ideally to connect intestinal flora, molecules, and host mucosa cells. It gives bacteria the capacity to inhabit ecological niches, communicate with host cells, and withstand environmental stresses. This study intends to evaluate the connection between GI tract biofilms and chronic mucosa diseases such as chronic gastritis, inflammatory bowel disease, and colorectal cancer. In each disease, we summarize the representative biofilm makers including Helicobacter pylori, adherent-invasive Escherichia coli, Bacteroides fragilis, and Fusobacterium nucleatum. We address biofilm's role in causing inflammation and the pro-carcinogenic stage in addition to discussing the typical resistance, persistence, and recurrence mechanisms seen in vitro. Biofilms may serve as a new biomarker for endoscopic and pathologic detection of gastrointestinal disease and suppression, which may be a useful addition to the present therapy strategy.

vicR overexpression in Streptococcus mutans causes aggregation and affects interspecies competition.

Streptococcus mutans is considered to be a major causative agent of dental caries. VicRK is a two-component signal transduction system (TCSTS) of S. mutans, which can regulate the virulence of S. mutans, such as biofilm formation, exopolysaccharide production, acid production, and acid resistance. Meanwhile, it can also regulate the production of mutacins (nlmC) through the TCSTS ComDE. In this study, we found that the vicR-overexpressing strain was more likely to aggregate to form cell clusters, leading to the formation of abnormal biofilm; the overexpression of vicR increased the length of the chain of S. mutans. Furthermore, the expression of the mutacins in the vicR overexpression strain was increased under aerobic conditions. Compared with the control strain and the parental strain, the vicR overexpression strain was more competitive against Streptococcus gordonii. But there was no significant difference against Streptococcus sanguinis. In clinical strains, the expression level of vicR was positively correlated with their competitive ability against S. gordonii. Transcriptional profiling revealed 24 significantly upregulated genes in the vicR-overexpressing strain, including nlmA, nlmB, nlmC, and nlmD encoding mutacins. Electrophoretic mobility shift assays and DNase I footprinting assays confirmed that VicR can directly bind to the promoter sequence of nlmD. Taken together, our findings further demonstrate that VicRK, an important TCSTS of S. mutans, is involved in S. mutans cell morphology and biofilm formation. VicRK regulates the production of more mutacins in S. mutans in response to oxygen stimulation. VicR can bind to the promoter sequence of nlmD, thereby directly regulating the production of mutacins NlmD.

Effect of Panax quinquefolius extract on Mycobacterium abscessus biofilm formation.

Mycobacterium abscessus (M. abscessus) can exist either as planktonic bacteria or as a biofilm. Biofilm formation is one of the important causes of conversion to resistance to antibiotics of bacteria that were previously sensitive when in their planktonic form, resulting in infections difficult to manage. Panax quinquefolius and its active ingredient ginsenosides have the potential ability in fighting pathogenic infections. In this study, the P. quinquefolius extract (PQE) showed good antibacterial/bactericidal activity against the M. abscessus planktonic cells. The extract reduced the biomass, thickness, and number of M. abscessus in the biofilm and altered its morphological characteristics as well as the spatial distribution of dead/alive bacteria. Moreover, the ginsenoside CK monomer had a similar inhibitory effect on M. abscessus planktonic bacteria and biofilm formation. Therefore, PQE and its monomer CK might be potential novel antimicrobial agents for the clinical prevention and treatment of M. abscessus, including biofilms in chronic infections.

Network pharmacology- and molecular simulation-based exploration of therapeutic targets and mechanisms of heparin for the treatment of sepsis/COVID-19.

Critically infected patients with COVID-19 (coronavirus disease 2019) are prone to develop sepsis-related coagulopathy as a result of a robust immune response. The mechanism underlying the relationship between sepsis and COVID-19 is largely unknown. LMWH (low molecular weight heparin) exhibits both anti-inflammatory and anti-coagulating properties that result in a better prognosis of severely ill patients with COVID-19 co-associated with sepsis-induced coagulopathy or with a higher D-dimer value. Heparin-associated molecular targets and their mechanism of action in sepsis/COVID-19 are not well understood. In this work, we characterize the pharmacological targets, biological functions and therapeutic actions of heparin in sepsis/COVID-19 from the perspective of network pharmacology. A total of 38 potential targets for heparin action against sepsis/COVID-19 and 8 core pharmacological targets were identified, including IL6, KNG1, CXCL8, ALB, VEGFA, F2, IL10 and TNF. Moreover, enrichment analysis showed that heparin could help in treating sepsis/COVID-19 through immunomodulation, inhibition of the inflammatory response, regulation of angiogenesis and antiviral activity. The pharmacological effects of heparin against these targets were further confirmed by molecular docking and simulation analysis, suggesting that heparin exerts effective binding capacity by targeting the essential residues in sepsis/COVID-19. Prospective clinical practice evaluations may consider the use of these key prognostic indicators for the treatment of sepsis/COVID-19.Communicated by Ramaswamy H. Sarma.

Combined transcriptomic and metabolomic analysis of Salmonella in the presence or absence of PhoP-PhoQ system under low Mg2+ conditions.

INTRODUCTION: Previous reports revealed the role played by Salmonella PhoP-PhoQ system in virulence activation, antimicrobial tolerance and intracellular survival, the impact of PhoP-PhoQ on cell metabolism has been less extensively described. OBJECTIVES: The aim of this study is to address whether and how the PhoP-PhoQ system affects the cell metabolism of Salmonella. METHODS: We constructed a Salmonella phoP deletion mutant strain TT-81 (PhoP-OFF), a Salmonella PhoP constitutively expressed strain TT-82 (PhoP-ON) and a wild-type Salmonella PhoP strain TT-80 (PhoP-N), using P22-mediated generalized transduction or λ Red-mediated targeted mutagenesis. We then measured the in vitro growth kinetics of all test strains and determined their metabolomic and transcriptomic profiles using gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) and RNA-seq technique, respectively. RESULTS: Low-Mg2+ conditions impaired the growth of the phoP deletion mutant strain TT-81 (PhoP-OFF) dramatically. 42 metabolites in the wild-type PhoP strain TT-80 (PhoP-N) and 28 metabolites in the PhoP constitutively expressed strain TT-82 (PhoP-ON) changed by the absence of phoP. In contrast, the level of 19 compounds in TT-80 (PhoP-N) changed comparing to the PhoP constitutively expressed strain TT-82 (PhoP-N). The mRNA level of 95 genes in TT-80 (PhoP-N) changed when phoP was disrupted, wherein 78 genes downregulated and 17 genes upregulated. 106 genes were determined to be differentially expressed between TT-81 (PhoP-OFF) and TT-82 (PhoP-ON). While only 16 genes were found to differentially expressed between TT-82 (PhoP-ON) and TT-80 (PhoP-N). CONCLUSION: Our findings confirmed the impact of PhoP-PhoQ system on lipopolysaccharide (LPS) modification, energy metabolism, and the biosynthesis or transport of amino acids. Most importantly, we demonstrated that the turnover of a given metabolite could respond differentially to the level of phoP. Taken together, the present study provided new insights into the adaptation of Salmonella to the host environment and helped to characterize the impact of the PhoP-PhoQ system on the cell metabolism.

Discovery and Mechanistic Study of Mycobacterium tuberculosis PafA Inhibitors.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb) and is ranked as the second killer infectious disease after COVID-19. Proteasome accessory factor A (PafA) is considered an attractive target because of its low sequence conservation in humans and its role in virulence. In this study, we designed a mutant of Mtb PafA that enabled large-scale purification of active PafA. Using a devised high-throughput screening assay, two PafA inhibitors were discovered. ST1926 inhibited Mtb PafA by binding in the Pup binding groove, but it was less active against Corynebacterium glutamicum PafA because the ST1926-binding residues are not conserved. Bithionol bound to the conserved ATP-binding pocket, thereby, inhibits PafA in an ATP-competitive manner. Both ST1926 and bithionol inhibited the growth of an attenuated Mtb strain (H37Ra) at micromolar concentrations. Our work thus provides new tools for tuberculosis research and a foundation for future PafA-targeted drug development for treating tuberculosis.

Improved Conventional and New Approaches in the Diagnosis of Tuberculosis.

Tuberculosis (TB) is a life-threatening infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). Timely diagnosis and effective treatment are essential in the control of TB. Conventional smear microscopy still has low sensitivity and is unable to reveal the drug resistance of this bacterium. The traditional culture-based diagnosis is time-consuming, since usually the results are available after 3-4 weeks. Molecular biology methods fail to differentiate live from dead M. tuberculosis, while diagnostic immunology methods fail to distinguish active from latent TB. In view of these limitations of the existing detection techniques, in addition to the continuous emergence of multidrug-resistant and extensively drug-resistant TB, in recent years there has been an increase in the demand for simple, rapid, accurate and economical point-of-care approaches. This review describes the development, evaluation, and implementation of conventional diagnostic methods for TB and the rapid new approaches for the detection of M. tuberculosis.

Deletion of the yqeK gene leads to the accumulation of Ap4A and reduced biofilm formation in Streptococcus mutans.

Diadenosine-5',5'''-P1, P4-tetraphosphate (Ap4A) is a second messenger playing a crucial role in various life activities of bacteria. The increase of Ap4A expression is pleiotropic, resulting in an impairment in the formation of biofilm and other physiological functions in some bacteria. However, Ap4A function in Streptococcus mutans, an important pathogen related to dental caries, remains unknown. In this work, the Ap4A hydrolase, YqeK, was identified and characterized in S. mutans. Then, the effects of yqeK deletion on the growth, biofilm formation, and exopolysaccharide (EPS) quantification in S. mutans were determined by the assessment of the growth curve, crystal violet, and anthrone-sulfuric acid, respectively, and visualized by microscopy. The results showed that the in-frame deletion of the yqeK gene in S. mutans UA159 led to an increase in Ap4A levels, lag phase in the early growth, as well as decrease in biofilm formation and water-insoluble exopolysaccharide production. Global gene expression profile showed that the expression of 88 genes was changed in the yqeK mutant, and among these, 42 were upregulated and 46 were downregulated when compared with the wild-type S. mutans UA159. Upregulated genes were mainly involved in post-translational modification, protein turnover, and chaperones, while downregulated genes were mainly involved in carbohydrate transport and metabolism. Important virulence genes related to biofilms, such as gtfB, gtfC, and gbpC, were also significantly downregulated. In conclusion, these results indicated that YqeK affected the formation of biofilms and the expression of biofilm-related genes in S. mutans.

Anti-tuberculosis drug development via targeting the cell envelope of Mycobacterium tuberculosis.

Mycobacterium tuberculosis possesses a dynamic cell envelope, which consists of a peptidoglycan layer, a mycolic acid layer, and an arabinogalactan polysaccharide. This envelope possesses a highly complex and unique structure representing a barrier that protects and assists the growth of M. tuberculosis and allows its adaptation to the host. It regulates the immune response of the host cells, causing their damage. Therefore, the cell envelope of M. tuberculosis is an attractive target for vaccine and drug development. The emergence of multidrug-resistant as well as extensively drug resistant tuberculosis and co-infection with HIV prevented an effective control of this disease. Thus, the discovery and development of new drugs is a major keystone for TB treatment and control. This review mainly summarizes the development of drug enzymes involved in the biosynthesis of the cell wall in M. tuberculosis, and other potential drug targets in this pathway, to provide more effective strategies for the development of new drugs.

Mycobacterium tuberculosis VapC4 toxin engages small ORFs to initiate an integrated oxidative and copper stress response.

The Mycobacterium tuberculosis (Mtb) VapBC4 toxin-antitoxin system is essential for the establishment of Mtb infection. Using a multitier, systems-level approach, we uncovered the sequential molecular events triggered by the VapC4 toxin that activate a circumscribed set of critical stress survival pathways which undoubtedly underlie Mtb virulence. VapC4 exclusively inactivated the sole transfer RNACys (tRNACys) through cleavage at a single site within the anticodon sequence. Depletion of the pool of tRNACys led to ribosome stalling at Cys codons within actively translating messenger RNAs. Genome mapping of these Cys-stalled ribosomes unexpectedly uncovered several unannotated Cys-containing open reading frames (ORFs). Four of these are small ORFs (sORFs) encoding Cys-rich proteins of fewer than 50 amino acids that function as Cys-responsive attenuators that engage ribosome stalling at tracts of Cys codons to control translation of downstream genes. Thus, VapC4 mimics a state of Cys starvation, which then activates Cys attenuation at sORFs to globally redirect metabolism toward the synthesis of free Cys. The resulting newly enriched pool of Cys feeds into the synthesis of mycothiol, the glutathione counterpart in this pathogen that is responsible for maintaining cellular redox homeostasis during oxidative stress, as well as into a circumscribed subset of cellular pathways that enable cells to defend against oxidative and copper stresses characteristically endured by Mtb within macrophages. Our ability to pinpoint activation or down-regulation of pathways that collectively align with Mtb virulence-associated stress responses and the nonreplicating persistent state brings to light a direct and vital role for the VapC4 toxin in mediating these critical pathways.

Inhibitory effects of sodium new houttuyfonate on growth and biofilm formation of Streptococcus mutans.

The present study aimed to assess the impact of sodium new houttuyfonate (SNH) on growth and biofilm formation of Streptococcus mutans, and the combinatorial effects of SNH with cariostatic agents. The effects of SNH on S. mutans planktonic cultures were assessed by growth curve assay. The effects of SNH on S. mutans biofilm and extracellular polysaccharides (EPS) production were observed via crystal violet (CV) assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, colony-forming unit (CFU) counting assay, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Quantitative real-time polymerase chain reaction (qPCR) was applied to investigate the regulatory effects of SNH on the expression of virulence genes of S. mutans. Checkerboard microdilution assay was performed to investigate the combinatorial effects of SNH with two common cariostatic agents. SNH acted as an inhibitor on planktonic cell growth, biofilm formation and EPS production of S. mutans. SNH also downregulated the expression of gtfBCD and comDE systems and exhibited synergism with chlorhexidine (CHX). In conclusion, this study indicated a possibility for SNH to become an anticaries agents by its antimicrobial activity and synergistic effects with CHX against S. mutans.

Ursolic acid inhibits multi-species biofilms developed by Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii.

OBJECTIVE: The current study aimed to assess the antimicrobial activity of ursolic acid (UA) against multi-species biofilms formed by Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii, as well as to measure its biocompatibility. METHODS: Crystal violet staining, CFU counting, CCK-8 assays and scanning electron microscopy (SEM) were applied to investigate the effect of UA on multi-species biofilms. UA's effect on exopolysaccharides (EPS) production was measured using confocal laser scanning microscopy (CLSM) and the anthrone-sulfuric acid method. Fluorescent in situ hybridization (FISH) was applied to visualize and quantify the microbial composition of multi-species biofilms. Quantitative real-time PCR (qRT-PCR) was used to measure the expression of virulence genes of S. mutans, S. sanguinis, and S. gordonii under UA treatment. Moreover, CCK-8 assays were performed to evaluate its cytotoxicity against human oral keratinocytes (HOKs) and human gingival epithelial cells (HGEs). RESULTS: The results showed that UA had significant antimicrobial activity against common oral streptococci. UA also reduced the EPS synthesis of oral streptococci and suppressed gtf genes' expression. In addition, UA reduced the proportion of S. mutans in multi-species biofilms. Besides, UA had low cytotoxicity against HOKs and HGEs. CONCLUSIONS: UA exhibited antibiofilm activity against oral pathogenic bacteria and had the potential to be used in dental caries treatment.

Shared bicycle microbial community: a potential antibiotic-resistant bacteria warehouse.

Shared bicycle is an emerging form of public transportation in China and around the world. However, the bacterial community and drug-resistant microbiome on these bicycles have not been reported. Samples from 10 shared bicycles were observed by scanning electron microscopy (SEM). Nine samples collected from 90 shared bicycles in three different kinds of location (hospital, metro station, shopping mall) were used for full-length 16S rDNA gene analysis to figure out the bacterial composition of the shared bicycle. Samples from 32 shared bicycles were used to investigate culturable drug-resistant bacteria of the shared bicycle bacterial community. It was found that in the shared bicycle bacterial community, Bacillus was the most abundant bacteria, as determined by both SEM observation and full-length 16S rDNA gene analysis. For the analysis of drug-resistant bacteria, Bacillus showed the strongest drug resist ability. Moreover, the resistances to bacitracin and sulfamethoxazole were the most common among all types of bacteria. Our study provides an important reference for the prevention of the potential spread of drug-resistant bacteria through shared bicycles.

Quantitative acetylome analysis reveals involvement of glucosyltransferase acetylation in Streptococcus mutans biofilm formation.

Streptococcus mutans (S. mutans) effectively utilizes dietary sucrose for the exopolysaccharide productions, which are mostly synthesized by the effects of glucosyltransferases (Gtfs). In the present study, the acetylome of S. mutans was identified and quantitative acetylome analysis of the bacterial biofilm growth (SMB) was compared with that of planktonic growth (SMP). The dynamic changes of protein acetylation were quantified using the integrated approach involving TMT labeling and Kac affinity enrichment followed by high-resolution mass spectrometry-based quantitative proteomics. In total, 973 acetylation sites in 445 proteins were identified, among which 617 acetylation sites in 302 proteins were quantitated. The overall analysis indicated that 22.7% of proteins were acetylated. Among the quantified proteins in SMB, the acetylation degree of lysine in 56 sites increased, while that of lysine decreased in 52 sites. In the acetylome of S. mutans, six significantly enriched motifs were identified and obtained including Kac****K, KacF, Kac****R, KacY, KacH, F*Kac. In addition, KEGG pathway-based enrichment analysis indicated significant enrichments in glycolysis/gluconeogenesis, and RNA degradation. Particularly, most downregulated acetylated lysine proteins were glucosyltransferase-SI, glucosyltransferase-I, and glucosyltransferase-S in S. mutans biofilm, which probably reveals a switch-off mechanism for the regulation of glucosyltransferases function during the biofilm development.