ompX contribute to biofilm formation, osmotic response and swimming motility in Citrobacter werkmanii.

Citrobacter werkmanii, an aerobe and mesophilic Proteobacterium, is universal in industrial putrefaction, coastal water, and human blood. Our previous studies have discovered that outer membrane protein X (OmpX) of C. werkmanii is involved in calcium response, but the underlying mechanisms and its molecular characteristics remain elusive. To that end, the ompX gene was deleted from the genome of C. werkmanii and its phenotypic variations were thoroughly investigated in conjunction with the wild type (WT) and complementary strains using biochemical and molecular techniques such as RNA-Seq, respectively. The results demonstrated that deleting ompX reduces biofilm formation on polystyrene and glass surfaces. Meanwhile, ΔompX's swimming ability but not for its twitching or swarming abilities, was also reduced on semi-solid plates compared with WT, which was caused by inhibition of flagellar assembly genes, such as flgC, flhB, and fliE, etc. Furthermore, ompX inactivation altered susceptibility to various bactericide classes, as well as responses to Ca2+ and Mg2+ stress. In addition, when compared to WT, ΔompX captures a total of 1,357 deferentially expressed genes (DEGs), of which 465 were up-regulated and 892 were down-regulated, which can be enriched into various GO ontology and KEGG pathway terms. Furthermore, ompX, as well as ompD and ompW, can be modulated at the transcriptional levels by rbsR and tdcA. Overall, the ompX gene contributed to a variety of biological functions in C. werkmanii and could be served as a targeted site for controlling biofilm formation and developing new bactericides.

Proteomic signatures of synergistic interactions in antimicrobials.

Mounting evidence has shown that antimicrobial agents can interfere synergistically with bacterial viability and proliferation when acting together at both the planktonic and biofilm levels without clear underlying molecular mechanisms. Here, multiplexed proteomics by iTRAQ was used to study the interplay between two biocides, the isothiazolone 1,2-benzisothiazolin-3-one (BIT) and the chelating agent disodium ethylenediaminetetraacetic acid (EDTA-2Na), employing the Citrobacter werkmanii as a model system. We first confirmed that these two biocides act synergistically on this bacterial species and then extracted the proteomic profiles of C. werkmanii cells in the presence of BIT, EDTA-2Na, and their combinations. In particular, we identified 43 core proteins that are differentially expressed in all three conditions simultaneously. Meanwhile, we found that these core proteins are consistently up-regulated when these two biocides are present, but not for single biocides, where we found a balanced mix of up- and down-regulation. Meanwhile, most of the deletion mutants of the core DEPs exhibited biofilm growth inhibition under joint biocide action, while their response was very heterogenous, with respect to the wild-type strain. Together, our results show that while BIT and EDTA-2Na act on multiple protein targets, they interact synergistically at the protein level in a very consistent manner. SIGNIFICANCE: Our preliminary experiments have demonstrated that a combination of 1,2-benzisothiazolin-3-one (BIT) and EDTA-2Na shows higher inhibitory effects on planktonic growth and biofilm formation in both C. werkmanii and Staphylococcus aureus than when these two biocides act alone. However, the mechanistic basis of such synergistic interaction is still unknown. Therefore, the key proteins involved in the above-mentioned enhanced antimicrobial synergy were elucidated using multiplexed proteomics analysis by isobaric tags for relative and absolute quantification (iTRAQ). Our results reveal that the joint action of BIT and EDTA-2Na induces consistent protein expression alteration in a set of core proteins of C. werkmanii, which underlies a strong synergistic antimicrobial effect, which increase our understanding of the action modes of BIT and EDTA-2Na as well as their combinations.

Outer membrane protein of OmpF contributes to swimming motility, biofilm formation, osmotic response as well as the transcription of maltose metabolic genes in Citrobacter werkmanii.

Bacterial outer membrane proteins (Omps) are essential for environmental sensing, stress responses, and substance transport. Our previous study discovered that OmpA contributes to planktonic growth, biocide resistance, biofilm formation, and swimming motility in Citrobacter werkmanii, whereas the molecular functions of OmpF in this strain are largely unknown. Thus, in this study, the ompF gene was firstly knocked out from the genome of C. werkmanii using a homologous recombination method, and its phenotypical alternations of ∆ompF were then thoroughly characterized using biochemical and molecular approaches with the parental wild type (WT) and complementary (∆ompF-com) strains. The results demonstrated that the swimming ability of ∆ompF on semi-solid plates was reduced compared to WT due to the down-regulation of flgC, flgH, fliK, and fliF. Meanwhile, ompF deletion reduces biofilm formation on both glass and polystyrene surfaces due to decreased cell aggregation. Furthermore, ompF inactivation induced different osmotic stress (carbon sources and metal ions) responses in its biofilms when compared to WT and ∆ompF-com. Finally, a total of 6 maltose metabolic genes of lamB, malE, malK, malG, malM, and malF were all up-regulated in ∆ompF. The gene knockout and HPLC results revealed that the MalEFGK2 cluster was primarily responsible for maltose transport in C. werkmanii. Furthermore, we discovered for the first time that the upstream promoter of OmpF and its transcription can be combined with and negatively regulated by MalT. Overall, OmpF plays a role in a variety of biochemical processes and molecular functions in C. werkmanii, and it may even act as a targeted site to inhibit biofilm formation.

Roles of ompA of Citrobacter werkmanii in bacterial growth, biocide resistance, biofilm formation and swimming motility.

The genus Citrobacter is commonly found in environmental and industrial settings, some members of which have been used for bioremediation of heavy metals owing to the absorption ability of their biofilms. Although our previous studies have found that the outer membrane protein A (OmpA) contributes to the process of Citrobacter werkmanii biofilm formation, the underlying mechanisms remain elusive. Therefore, we deleted ompA from the genome of C. werkmanii and investigated its phenotypes in comparison to the wild type strain (WT) and the complementary strain using biochemical and molecular techniques including RNA-Seq. Our results demonstrated that the deletion of ompA led to an increase in biofilm formation on both polystyrene and glass surfaces due to upregulation of some biofilm formation related genes. Meanwhile, swimming ability, which is mediated by activation of flagellar assembly genes, was increased on semi-solid plates in the ∆ompA strain when compared with WT. Additionally, inactivation of ompA also caused increased 1,2-benzisothiazolin-3-one (BIT) resistance, differential responses to Ca2+ stress, curli protein expression and cellulose production. Finally, ∆ompA caused differential expression of a total of 1470 genes when compared with WT, of which 146 were upregulated and 1324 were downregulated. These genes were classified into different Gene Ontology (GO) and KEGG pathways. In summary, ompA in C. werkmanii contributes to a variety of biological functions and may act as a target site to modulate biofilm formation. KEY POINTS: • ompA is a negative regulator for biofilm formation by C. werkmanii. • ompA inhibits swimming motility of C. werkmanii. • ompA deletion causes different expression profiles in C. werkmanii.

Chemical Components of Aqueous Extracts of Melia azedarach Fruits and Their Effects on The Transcriptome of Staphylococcus aureus.

Staphylococcus aureus is the causative agent of numerous and varied clinical infections. Crude aqueous extracts of Melia azedarach fruits inhibit the planktonic growth and initial biofilm formation of S. aureus in a dose-dependent manner. Moreover, the biofilm topologies became sparse and decreased as the concentration of the aqueous extracts increased. RNA-Seq analyses revealed 532 differentially expressed genes (DEGs) after S. aureus exposure to 0.25 g/ml extracts; 319 of them were upregulated, and 213 were downregulated. The majority of DEGs were categorized into abundant sub-groups in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Finally, untargeted UHPLC-MS/MS analyses of the aqueous extracts of M. azedarach fruits demonstrated a highly complex profile in positive and negative electrospray ionization modes. The extracts primarily consisted of lipids and lipid-like molecules, organic acids and their derivatives, phenylpropanoids, polyketides, organoheterocyclic compounds, and benzenoids annotated by abundant lipid maps and KEGG pathways. Overall, this study provides evidences that the aqueous extracts of M. azedarach fruits can control S. aureus infections and sought to understand the mode of action of these extracts on S. aureus.

Diallyl disulfide from garlic oil inhibits Pseudomonas aeruginosa virulence factors by inactivating key quorum sensing genes.

Garlic oil can disrupt the quorum sensing (QS) pathways of the opportunistic pathogen Pseudomonas aeruginosa; however, the underlying mechanisms for this effect are unclear. Diallyl disulfide (DADS) is one of the most abundant sulfur-containing compounds in garlic oil. This study investigated the effects of DADS on the growth, virulence factor production (elastase, pyocyanin, biofilm, and swarming motility), and essential gene expression of P. aeruginosa PAO1, particularly as they apply to QS and virulence. DADS at 1.28 mg/mL did not affect P. aeruginosa PAO1 growth, although it decreased elastase and pyocyanin production, biofilm formation, and swarming motility. Each of these phenomena is regulated by the three QS systems of P. aeruginosa PAO1 (las, rhl, and pqs). Real-time q-PCR revealed that DADS down-regulated the transcription levels of several important QS genes (lasI, lasR, rhlI, rhlR, pqsA, and pqsR) in the three systems. Furthermore, the transcription levels of QS-regulated virulence genes were also down-regulated. The lasB gene, encoding LasB elastase, is co-regulated by the las, rhl, and pqs systems, and thus the down-regulation of genes across the three systems further down-regulated lasB. Additionally, phzM (encoding pyocyanin), pslB (responsible for the production of a biofilm matrix polysaccharide), and chiC (encoding chitinase) were positively activated by LasR, and a decrease in lasR transcription further down-regulated the transcription of phzM, pslB, and chiC. Hence, DADS inhibits P. aeruginosa PAO1 virulence factors by inactivating the transcription of key genes across three different QS systems.

Diallyl Disulfide From Garlic Oil Inhibits Pseudomonas aeruginosa Quorum Sensing Systems and Corresponding Virulence Factors.

Previously, we determined that diallyl disulfide (DADS) from garlic oil can inhibit Pseudomonas aeruginosa PAO1 pathogenic factors by inactivating the transcription of key genes from three quorum sensing (QS) systems (las, rhl, and pqs) based on the effects of DADS on growth, virulence factor production (elastase, pyocyanin, biofilm, and swarming motility), and RNA transcription (real-time q-PCR). To further investigate the mechanisms underlying the inhibition of the three P. aeruginosa QS systems by DADS, high-throughput RNA and proteome sequencing techniques were used to study differences in the transcriptional and proteome expression of P. aeruginosa PAO1 following treatment with DADS. The RNA-seq and proteomic data are available via NCBI Gene Expression Omnibus database with accession number GSE118801 and ProteomeXchange with identifier PXD011144, respectively. The experimental results indicated that all key genes of the three QS systems (las, rhl, and pqs) of P. aeruginosa PAO1 as well as the virulence factors (including exoprotease LasA, elastase LasB, lectin LecA and LecB, pyocyanin biosynthesis, and biofilm formation) regulated by these three QS systems were inhibited. This is consistent with our previous studies on the physiology, biochemistry, and RNA expression of P. aeruginosa treated with DADS. Additionally, our results also indicated that bacterial motility, chemotaxis, and two-component systems were inhibited by DADS treatment. All these changes abolish the sensitivity of P. aeruginosa PAO1 to environmental stimuli and cause the cells to be in a state of passivation. Further research is needed to determine how QS systems regulate these functions. Our findings could potentially contribute to the treatment and control of P. aeruginosa infection, virulence, and pathogenicity.

The dynamics and mechanism of the antimicrobial activity of tea tree oil against bacteria and fungi.

Tea tree oil (TTO) is a yellow liquid extracted from Melaleuca alternifolia. Although the antimicrobial activity of TTO has been known for a long time, its specific antimicrobial effects and mechanism underlying these remain poorly characterized. The present study investigated the chemical composition of TTO and the dynamics and mechanism of its antimicrobial activities in two bacterial and two fungal strains. Gas chromatography-mass spectrometry analysis identified alkenes and alcohols as the main constituents of TTO. Terpinen-4-ol was the most abundant individual component, accounting for approximately 23 % of the TTO. Poisoned food technique assessment showed that the minimum inhibitory concentrations of TTO for bacterial strains (Escherichia coli and Staphylococcus aureus) and fungal strains (Candida albicans and Aspergillus niger) were 1.08 and 2.17 mg/mL, respectively. Antimicrobial dynamic curves showed that with increasing concentrations of TTO, the rate of cell killing and the duration of growth lag phase increased correspondingly. These data indicated that TTO produced concentration and time-dependent antimicrobial effects. The minimum bactericidal and fungicidal concentrations of TTO were 2.17, 4.34, and 4.34 against E. coli, S. aureus, and C. albicans, respectively. However, A. niger conidia were not completely eradicated, even after 3 days in the presence of 17.34 mg/mL TTO. Transmission electron microscopy images indicated that TTO penetrated the cell wall and cytoplasmic membrane of all the tested bacterial and fungal strains. TTO may also penetrate fungal organelle membrane. These findings indicated that TTO maybe exerts its antimicrobial effects by compromising the cell membrane, resulting in loss of the cytoplasm and organelle damage, which ultimate leads to cell death.