Pilus Expression Research Articles

Diffusible signal factor (DSF)-mediated quorum sensing modulates swarming in Xanthomonas albilineans.

Xanthomonas spp. are plant pathogens known for significantly impacting crop yields. Among them, Xanthomonas albilineans (Xal) is notable for colonizing the xylem and causing sugarcane leaf scald disease. This study employed homologous recombination to mutate quorum sensing (QS) regulatory genes (rpf) to investigate their role in Xal pathogenicity. Deletions of rpfF (ΔrpfF), rpfC (ΔrpfC), and rpfG (ΔrpfG) led to reduced swarming, growth, and virulence. However, DSF supplementation restored swarming and growth in the ΔrpfF mutant. Deleting rpfC, rpfG, and rpfF also reduced twitching motility and affected Type IV Pilus (T4P) expression. Transcriptomic analysis revealed that ΔrpfF positively regulates flagellar genes. DSF supplementation in ΔrpfF (ΔrpfF-DSF) modulated the expression of flagellar, chemotaxis, and T4P genes. These findings elucidate the DSF-mediated swarming pathway in Xal and provide valuable insights into its regulatory mechanisms.

Evolutionary adaptation of KPC-2-producing Pseudomonas aeruginosa high-risk sequence type 463 in a lung transplant patient

ObjectivesKPC-2-producing Pseudomonas aeruginosa high-risk sequence type (ST) 463 is increasingly prevalent in China and poses severe threats to public health. In this study, we aimed to investigate within-host adaptive evolution of this clone during therapy. MethodsUsing nine serial respiratory isolates from a post-lung transplantation patient undergoing multiple antibiotic treatments, we conducted genomic, transcriptomic and phenotypic analyses to uncover the adaptive mechanisms of a KPC-2-producing ST463 P. aeruginosa strain. ResultsThe early-course isolates exhibited low-level resistance to ceftazidime/avibactam (CZA), facilitated by the blaKPC-2 gene's presence on both chromosome and plasmid, and its overexpression. Comparative genomic analysis revealed that chromosomal integration of blaKPC-2 resulted from intracellular replicative transposition of the plasmid-derived IS26-blaKPC-2-IS26 composite transposon. As the infection progressed, selective pressures, predominantly from antibiotic interventions and host immune response, led to significant genomic and phenotypic changes. The late-course isolates developed a Δ242-GT-243 deletion in plasmid-encoded blaKPC-2 (blaKPC-14) after sustained CZA exposure, conferring high-level CZA resistance. Increased expression of pili and extracellular polysaccharides boosted biofilm formation. A D143N mutation in the global regulator vfr rendered the strain aflagellate by abrogating the ability of fleQ to positively regulate flagellar gene expression. The enhancement of antibiotic resistance and immune evasion collaboratively facilitated the prolonged survival of ST463 P. aeruginosa within the host. ConclusionsOur findings highlight the remarkable capacity of ST463 P. aeruginosa in adapting to the dynamic host pressures, supporting its persistence and dissemination in healthcare.

Quorum Sensing Enhances Direct Interspecies Electron Transfer in Anaerobic Methane Production.

Direct interspecies electron transfer (DIET) provides an innovative way to achieve efficient methanogenesis, and this study proposes a new approach to upregulate the DIET pathway by enhancing quorum sensing (QS). Based on long-term reactor performance, QS enhancement achieved more vigorous methanogenesis with 98.7% COD removal efficiency. In the control system, methanogenesis failure occurred at the accumulated acetate of 7420 mg of COD/L and lowered pH of 6.04, and a much lower COD removal of 41.9% was observed. The more significant DIET in QS-enhancing system was supported by higher expression of conductive pili and the c-Cyts cytochrome secretion-related genes, resulting in 12.7- and 10.3-fold improvements. Moreover, QS enhancement also improved the energy production capability, with the increase of F-type and V/A-type ATPase expression by 6.3- and 4.2-fold, and this effect probably provided more energy for nanowires and c-Cyts cytochrome secretion. From the perspective of community structure, QS enhancement increased the abundance of Methanosaeta and Geobacter from 54.3 and 17.6% in the control to 63.0 and 33.8%, respectively. Furthermore, the expression of genes involved in carbon dioxide reduction and alcohol dehydrogenation increased by 0.6- and 7.1-fold, respectively. Taken together, this study indicates the positive effects of QS chemicals to stimulate DIET and advances the understanding of the DIET methanogenesis involved in environments such as anaerobic digesters and sediments.

Colonization of vancomycin-resistant Enterococcus faecium in human-derived colonic epithelium: unraveling the transcriptional dynamics of host-enterococcal interactions.

Enterococcus faecium is an opportunistic pathogen able to colonize the intestines of hospitalized patients. This initial colonization is an important step in the downstream pathogenesis, which includes outgrowth of the intestinal microbiota and potential infection of the host. The impact of intestinal overgrowth on host-enterococcal interactions is not well understood. We therefore applied a RNAseq approach in order to unravel the transcriptional dynamics of E. faecium upon co-culturing with human derived colonic epithelium. Co-cultures of colonic epithelium with a hospital-associated vancomycin resistant (vanA-type) E. faecium (VRE) showed that VRE resided on top of the colonic epithelium when analyzed by microscopy. RNAseq revealed that exposure to the colonic epithelium resulted in upregulation of 238 VRE genes compared to the control condition, including genes implicated in pili expression, conjugation (plasmid_2), genes related to sugar uptake, and biofilm formation (chromosome). In total, 260 were downregulated, including the vanA operon located on plasmid_3. Pathway analysis revealed an overall switch in metabolism to amino acid scavenging and reduction. In summary, our study demonstrates that co-culturing of VRE with human colonic epithelium promotes an elaborate gene response in VRE, enhancing our insight in host-E. faecium interactions, which might facilitate the design of novel anti-infectivity strategies.

Csu pili dependent biofilm formation and virulence of Acinetobacter baumannii

Acinetobacter baumannii has emerged as one of the most common extensive drug-resistant nosocomial bacterial pathogens. Not only can the bacteria survive in hospital settings for long periods, but they are also able to resist adverse conditions. However, underlying regulatory mechanisms that allow A. baumannii to cope with these conditions and mediate its virulence are poorly understood. Here, we show that bi-stable expression of the Csu pili, along with the production of poly-N-acetyl glucosamine, regulates the formation of Mountain-like biofilm-patches on glass surfaces to protect bacteria from the bactericidal effect of colistin. Csu pilus assembly is found to be an essential component of mature biofilms formed on glass surfaces and of pellicles. By using several microscopic techniques, we show that clinical isolates of A. baumannii carrying abundant Csu pili mediate adherence to epithelial cells. In addition, Csu pili suppressed surface-associated motility but enhanced colonization of bacteria into the lungs, spleen, and liver in a mouse model of systemic infection. The screening of c-di-GMP metabolizing protein mutants of A. baumannii 17978 for the capability to adhere to epithelial cells led us to identify GGDEF/EAL protein AIS_2337, here denoted PdeB, as a major regulator of Csu pili-mediated virulence and biofilm formation. Moreover, PdeB was found to be involved in the type IV pili-regulated robustness of surface-associated motility. Our findings suggest that the Csu pilus is not only a functional component of mature A. baumannii biofilms but also a major virulence factor promoting the initiation of disease progression by mediating bacterial adherence to epithelial cells.

Ex-vivo RNA expression analysis of vaccine candidate genes in COPD sputum samples

BackgroundChronic obstructive pulmonary disease (COPD) is a lung disease characterised by airflow-limiting inflammation and mucus production. Acute exacerbations are a major cause of COPD-related morbidity and mortality and are mostly associated with bacterial or viral infections. A vaccine targeting non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat), the main bacteria associated with exacerbations, was tested in a Phase 2 trial. We assessed “ex-vivo” expression of vaccine candidate and housekeeping genes pd, pe, pilA, gapA, ompP6 of NTHi, and uspA2, parE, polA of Mcat in sputum samples of COPD patients and determined whether expression of the vaccine candidate genes pd, pe, pilA (NTHi) and uspA2 (Mcat) differed between stable and exacerbation samples.MethodsA single-centre, prospective, observational cohort study was conducted where 123 COPD patients were seen on enrolment, followed monthly for 2 years, and reviewed after onset of acute exacerbations. We selected 69 patients with sputum samples positive for NTHi or Mcat by PCR during at least one stable and one exacerbation visit. mRNA was isolated from the sputum, and expression of NTHi and Mcat genes was analysed with RT-PCR. Statistical analyses compared mRNA concentrations between stable and exacerbation samples and in relationship to COPD severity and exacerbation frequency.ResultsThe vaccine candidate genes were variably expressed in sputum samples, suggesting they are expressed in the lung. Absolute and relative expression of all NTHi vaccine candidate genes and Mcat uspA2 were similar between exacerbation and stable samples. Expression of pd and pilA was slightly associated with the number of exacerbations in the year before enrolment, and uspA2 with the disease severity status at enrolment.ConclusionsThe NTHi-Mcat vaccine candidate genes were expressed in sputum samples, and each gene had a specific level of expression. No statistically significant differences in gene expression were detectable between stable and exacerbation samples. However, the history of COPD exacerbations was slightly associated with the expression of pd, pilA and uspA2.Trial registration NCT01360398 (https://www.clinicaltrials.gov)Graphical abstract

In vitro adhesion, pilus expression, and in vivo amelioration of antibiotic-induced microbiota disturbance by Bifidobacterium spp. strains from fecal donors

ABSTRACT Fecal microbiota transplantation (FMT) is used routinely to treat recurrent Clostridioides difficile infection (rCDI) and investigated as a treatment for numerous conditions associated with gut microbiota alterations. Metagenomic analyses have indicated that recipient colonization by donor bacteria may be associated with favorable clinical outcomes. Bifidobacteria are abundant gut commensals associated with health. We have previously demonstrated that Bifidobacterium strains transferred in FMT can colonize recipients in long term, at least for a year, and recovered such strains by cultivation. This study addressed in vitro adhesion and pilus gene expression of long-term colonizing Bifidobacterium strains from FMT donors as well as in vivo colonization and capability to ameliorate antibiotic-induced microbiota disturbance. RNA-Seq differential gene expression analysis showed that the strongly adherent B. longum strains DY_pv11 and DX_pv23 expressed tight adherence and sortase-dependent pilus genes, respectively. Two B. longum strains, adherent DX_pv23 and poorly adhering DX_pv18, were selected to address in vivo colonization and efficacy to restore antibiotic-disturbed microbiota in C57BL/6 murine model. DX_pv23 colonized mice transiently with a rate comparable to that of the B. animalis BB-12 used as a reference. Although long-term colonization was not observed with any of the three strains, 16S rRNA gene profiling revealed that oral administration of DX_pv23 enhanced the recovery of antibiotic-disturbed microbiota to the original configuration significantly better than the other strains. The findings suggest that selected strains from FMT donors, such as DX_pv23 in this study, may have therapeutic potential by in vitro expression of colonization factors and boosting endogenous gut microbiota.

FimH and Type 1 Pili Mediated Tumor Cell Cytotoxicity by Uropathogenic Escherichia coli In Vitro.

Uropathogenic Escherichia coli express hairlike proteinaceous surface projections, known as chaperone-usher pathway (CUP) pili. Type 1 pili are CUP pili with well-established pathogenic properties. The FimH adhesin subunit of type 1 pili plays a key role in the pathogenesis of urinary tract infections (UTIs) as it mediates the adhesion of the bacteria to urothelial cells of the bladder. In this study, two breast cancer cell lines, MDA-MB-231 and MCF-7, were used to demonstrate the cytotoxic activities of type 1 piliated uropathogenic E. coli UTI89 on breast cancer cells in a type 1 pili and FimH-mediated manner. E. coli were grown in static and shaking conditions to induce or inhibit optimal type 1 pili biogenesis, respectively. Deletion constructs of UTI89 ΔfimH and a complemented strain (UTI89 ΔfimH/pfimH) were further utilized to genetically assess the effect of type 1 pili and FimH on cancer cell viability. After incubation with the different strains, cytotoxicity was measured using trypan blue exclusion assays. UTI89 grown statically caused significant cytotoxicity in both breast cancer cell lines whereas cytotoxicity was reduced when the cells were incubated with bacteria grown under shaking conditions. The incubation of both MDA-MB-231 and MCF-7 with UTI89 Δfim operon or ΔfimH showed a significant reduction in cytotoxicity exerted by the bacterial strains, revealing that type 1 pili expression was necessary for cytotoxicity. Complementing the ΔfimH strain with pfimH reversed the phenotype, leading to a significant increase in cytotoxicity. Incubating type 1 pili expressing bacteria with the competitive FimH inhibitor D-mannose before cancer cell treatment also led to a significant reduction in cytotoxicity on both MDA-MB-231 and MCF-7 cancer cells, compared to vehicle control or D-mannose alone, indicating the requirement for functional FimH for cytotoxicity. Overall, our results reveal that, as opposed to UTI89 lacking type 1 pili, type 1 piliated UTI89 causes significant cancer cell mortality in a FimH-mediated manner, that is decreased with D-mannose.

A Single Residue within the MCR-1 Protein Confers Anticipatory Resilience.

The envelope stress response (ESR) of Gram-negative enteric bacteria senses fluctuations in nutrient availability and environmental changes to avert damage and promote survival. It has a protective role toward antimicrobials, but direct interactions between ESR components and antibiotic resistance genes have not been demonstrated. Here, we report interactions between a central regulator of ESR viz., the two-component signal transduction system CpxRA (conjugative pilus expression), and the recently described mobile colistin resistance protein (MCR-1). Purified MCR-1 is specifically cleaved within its highly conserved periplasmic bridge element, which links its N-terminal transmembrane domain with the C-terminal active-site periplasmic domain, by the CpxRA-regulated serine endoprotease DegP. Recombinant strains harboring cleavage site mutations in MCR-1 are either protease resistant or degradation susceptible, with widely differing consequences for colistin resistance. Transfer of the gene encoding a degradation-susceptible mutant to strains that lack either DegP or its regulator CpxRA restores expression and colistin resistance. MCR-1 production in Escherichia coli imposes growth restriction in strains lacking either DegP or CpxRA, effects that are reversed by transactive expression of DegP. Excipient allosteric activation of the DegP protease specifically inhibits growth of isolates carrying mcr-1 plasmids. As CpxRA directly senses acidification, growth of strains at moderately low pH dramatically increases both MCR-1-dependent phosphoethanolamine (PEA) modification of lipid A and colistin resistance levels. Strains expressing MCR-1 are also more resistant to antimicrobial peptides and bile acids. Thus, a single residue external to its active site induces ESR activity to confer resilience in MCR-1-expressing strains to commonly encountered environmental stimuli, such as changes in acidity and antimicrobial peptides. Targeted activation of the nonessential protease DegP can lead to the elimination of transferable colistin resistance in Gram-negative bacteria. IMPORTANCE The global presence of transferable mcr genes in a wide range of Gram-negative bacteria from clinical, veterinary, food, and aquaculture environments is disconcerting. Its success as a transmissible resistance factor remains enigmatic, because its expression imposes fitness costs and imparts only moderate levels of colistin resistance. Here, we show that MCR-1 triggers regulatory components of the envelope stress response, a system that senses fluctuations in nutrient availability and environmental changes, to promote bacterial survival in low pH environments. We identify a single residue within a highly conserved structural element of mcr-1 distal to its catalytic site that modulates resistance activity and triggers the ESR. Using mutational analysis, quantitative lipid A profiling and biochemical assays, we determined that growth in low pH environments dramatically increases colistin resistance levels and promotes resistance to bile acids and antimicrobial peptides. We exploited these findings to develop a targeted approach that eliminates mcr-1 and its plasmid carriers.

Pseudomonas aeruginosa Resists Phage Infection via Eavesdropping on Indole Signaling.

Phage therapy is challenged by the frequent emergence of bacterial resistance to phages. As an interspecies signaling molecule, indole plays important roles in regulating bacterial behaviors. However, it is unclear whether indole is involved in the phage-bacterium interactions. Here, we report that indole modulated phage resistance of Pseudomonas aeruginosa PAO1. Specifically, we found that the type IV pilus (T4P) acts as an important receptor for P. aeruginosa phages vB_Pae_S1 and vB_Pae_TR, and indole could protect P. aeruginosa against phage infection via decreasing the T4P-mediated phage adsorption. Further investigation demonstrated that indole downregulated the expression of genes pilA, pilB, and pilQ, which are essential for T4P assembly and activity. Indole inhibits phage attacks, but our data suggest that indole functions not through interfering with the AHL-based QS pathway, although las quorum sensing (QS) of P. aeruginosa PAO1 were reported to promote phage infection. Our finding confirms the important roles of indole in virus-host interactions, which will provide important enlightenment in promoting phage therapy for P. aeruginosa infections. IMPORTANCE Our finding is significant with respect to the study of the interactions between phage and host. Although the important roles of indole in bacterial physiology have been revealed, no direct examples of indole participating in phage-host interactions were reported. This study reports that indole could modulate the phage resistance of indole-nonproducing Pseudomonas aeruginosa PAO1 through inhibition of phage adsorption mechanism. Our finding will be significant for guiding phage therapy and fill some gaps in the field of phage-host interactions.

YihE is a novel binding partner of Rho and regulates Rho-dependent transcription termination in the Cpx stress response

The conjugative pilus expression (Cpx) stress response system can sense cell envelope stressors, such as misfolded proteins, and upregulate proteases to modify or degrade damaged proteins. YihE is a protein kinase implicated in the Cpx system, and Rho is a transcription termination factor in prokaryotes, but no functional connection between YihE and Rho has been reported. Here, we found that YihE can interact with Rho to form a binary complex with a stoichiometric ratio of 6:1 (Rho:YihE). A low resolution of Rho crystal structure in the presence of YihE was determined. YihE overexpression helped lessen the aberrant effects caused by Rho overexpression, including long cell morphology and other Rho-mediated phenotypes. Overall, YihE is a Rho binding partner that acts as a Rho antagonist in the Cpx stress. YihE binding to Rho would compete RNA from binding to Rho, thereby helping bacteria cope with stress through the regulation of Rho-dependent transcription termination.

Multi-cycle anaerobic digestion of hydrothermal liquefaction aqueous phase: Role of carbon and iron based conductive materials in inhibitory compounds degradation, microbial structure shaping, and interspecies electron transfer regulation

Anaerobic digestion is commonly performed to treat hydrothermal liquefaction aqueous phase (HTL-AP), but the conversion is limited by the inhibitory compounds. This study was conducted to investigate the effects of adding granular activated carbon, biochar, and magnetite on the digestion of HTL-AP. Moreover, the enriched aggregates and conductive materials were recovered after experiment, and then reused to study the effects of multi-cycle digestion on organics degradation, microbial activity, and methane production. Results showed that the conductive materials significantly contributed to the digestion performance and maintained high activity after recycling. The degradation of organic matters, especially the removal of toxic compounds, the enrichment of functional microbes, as well as the production of methane can be further enhanced with the reuse of conductive materials. Specifically, a 77% higher methane production rate was observed in magnetite assisted digester after recycling. More than 80% of the nitrogen organics were degraded with biochar, and all potential inhibitors were completely removed with GAC addition. The diversity and abundance of microbes were improved, and the dominant structure was established in the first batch, then the enrichment was further stabilized after the recycling. Meanwhile, the sludge conductivity was increased by 28.1 and 8.5 times in GAC and magnetite digesters in the first batch and was further enhanced by 8.0% and 16.4% after the recycling, indicating the improved expression of conductive pili and cytochromes of the microbes. These results point to the establishment of direct interspecies electron transfer. This study showed promising prospects for the utilization and recycling of conductive materials in the anaerobic digestion of HTL-AP and its long-term applications.

Characterization of M-Type-Specific Pilus Expression in Group A Streptococcus.

In addition to providing a typing mechanism for group A Streptococcus (GAS) isolates (T typing), cell surface pilus production impacts GAS virulence characteristics, including adherence and immune evasion. The pilus biosynthesis genes are located in the fibronectin- and collagen-binding T-antigen (FCT) region of the genome, and nine different FCT types, encoding more than 20 different T types, have been described. GAS isolates are not uniform in their degree or pattern of pilus expression, as highlighted by pilus production being thermoregulated in isolates that harbor the FCT-type FCT-3 (e.g., M-types M3 and M49) but not in isolates that harbor FCT-2 (e.g., M-type M1). Here, we investigated the molecular basis underlying our previous finding that M3 GAS isolates produce pili in lower abundance than M1 or M49 isolates do. We discovered that, at least in part, the low pilus expression observed for M3 isolates is a consequence of the repression of pilus gene expression by the CovR/CovS two-component regulatory system and of an M3-specific mutation in the nra gene, encoding a positive regulator of pilus gene expression. We also discovered that the orthologous transcriptional regulators RofA and Nra, whose encoding genes are located within FCT-2 and FCT-3, respectively, are not functionally identical. Finally, we sequenced the genome of an M3 isolate that had naturally undergone recombinational replacement of the FCT region, changing the FCT and T types of this strain from FCT-3/T3 to FCT-2/T1. Our study furthers the understanding of strain- and type-specific variation in virulence factor production by an important human pathogen. IMPORTANCE Our ability to characterize how a pathogen infects and causes disease, and consequently our ability to devise approaches to prevent or attenuate such infections, is inhibited by the finding that isolates of a given pathogen often show phenotypic variability, for example, in their ability to adhere to host cells through modulation of cell surface adhesins. Such variability is observed between isolates of group A Streptococcus (GAS), and this study investigates the molecular basis for why some GAS isolates produce pili, cell wall-anchored adhesins, in lower abundance than other isolates do. Given that pili are being considered as potential antigens in formulations of future GAS vaccines, this study may inform vaccine design.

OxyR contributes to virulence of Acidovorax citrulli by regulating anti-oxidative stress and expression of flagellin FliC and type IV pili PilA.

In many bacteria, OxyR acts as a transcriptional regulator that facilitates infection via degrading hydrogen peroxide (H2O2) generated by the host defense response. Previous studies showed that OxyR also plays an important role in regulating biofilm formation, cell motility, pili relate-genes expression, and surface polysaccharide production. However, the role of OxyR has not been determined in Acidovorax citrulli strain xjl12. In the current study, the qRT-PCR and western blot assays revealed that the expression level of oxyR was significantly induced by H2O2. The oxyR deletion mutant of A. citrulli was significantly impaired bacterial tolerance to oxidative stress and reduced catalase (CAT) activity. In addition, oxyR mutant resulted in reduced swimming motility, twitching motility, biofilm formation, virulence, and bacterial growth in planta by significantly affecting flagellin and type IV pili-related gene (fliC and pilA) expression. The qRT-PCR assays and western blot revealed that OxyR positively regulated the expression of fliC and pilA. Furthermore, bacterial one-hybrid assay demonstrated that OxyR directly affected pilA and fliC promoter. Through bacterial two-hybrid assay, it was found that OxyR can directly interact with PilA and FliC. These results suggest that OxyR plays a major role in the regulating of a variety of virulence traits, and provide a foundation for future research on the global effects of OxyR in A. citrulli.

Virulence Characteristics and Emerging Therapies for Biofilm-Forming Acinetobacter baumannii: A Review.

Simple SummaryAcinetobacter baumannii (A. baumannii) is one of the ESKAPE organisms and has the competency to build biofilms. These biofilms account for the most nosocomial infections all over the world. This review reflects on the various physicochemical and environmental factors such as adhesion, pili expression, growth surfaces, drug-resistant genes, and virulence factors that profoundly affect its resistant forte. Emerging drug-resistant issues and limitations to newer drugs are other factors affecting the hospital environment. Here, we discuss newer and alternative methods that can significantly enhance the susceptibility to Acinetobacter spp. Many new antibiotics are under trials, such as GSK-3342830, The Cefiderocol (S-649266), Fimsbactin, and similar. On the other hand, we can also see the impact of traditional medicine and the secondary metabolites of these natural products’ application in searching for new treatments. The field of nanoparticles has demonstrated effective antimicrobial actions and has exhibited encouraging results in the field of nanomedicine. The use of various phages such as vWUPSU and phage ISTD as an alternative treatment for its specificity and effectiveness is being investigated. Cathelicidins obtained synthetically or from natural sources can effectively produce antimicrobial activity in the micromolar range. Radioimmunotherapy and photodynamic therapy have boundless prospects if explored as a therapeutic antimicrobial strategy.Acinetobacter species is one of the most prevailing nosocomial pathogens with a potent ability to develop antimicrobial resistance. It commonly causes infections where there is a prolonged utilization of medical devices such as CSF shunts, catheters, endotracheal tubes, and similar. There are several strains of Acinetobacter (A) species (spp), among which the majority are pathogenic to humans, but A. baumannii are entirely resistant to several clinically available antibiotics. The crucial mechanism that renders them a multidrug-resistant strain is their potent ability to synthesize biofilms. Biofilms provide ample opportunity for the microorganisms to withstand the harsh environment and further cause chronic infections. Several studies have enumerated multiple physiological and virulence factors responsible for the production and maintenance of biofilms. To further enhance our understanding of this pathogen, in this review, we discuss its taxonomy, pathogenesis, current treatment options, global resistance rates, mechanisms of its resistance against various groups of antimicrobials, and future therapeutics.

Actinomycin D: a novel Pseudomonas aeruginosa quorum sensing inhibitor from the endophyte Streptomyces cyaneochromogenes RC1.

The infections caused by Pseudomonas aeruginosa are difficult to treat due to its multidrug resistance. A promising strategy for controlling P. aeruginosa infection is targeting the quorum sensing (QS) system. Actinomycin D isolated from the metabolite of endophyte Streptomyces cyaneochromogenes RC1 exhibited good anti-QS activity against P. aeruginosa PAO1. Actinomycin D (50, 100, and 200μg/mL) significantly inhibited the motility as well as reduced the production of multiple virulence factors including pyocyanin, protease, rhamnolipid, and siderophores. The images of confocal laser scanning microscopy and scanning electron microscopy revealed that the treatment of actinomycin D resulted in a looser and flatter biofilm structure. Real-time quantitative PCR analysis showed that the expression of QS-related genes lasI, rhlI, rhlR, pqsR, pslA, and pilA were downregulated dramatically. The production of QS signaling molecules N-(3-oxododecanoyl)-L-homoserine lactone and N-butanoyl-L-homoserine lactone were also decreased by actinomycin D. These findings suggest that actinomycin D, a potent in vitro anti-virulence agent, is a promising candidate to treat P. aeruginosa infection by interfering with the QS systems.

The Role of Exopolysaccharides in Direct Interspecies Electron Transfer.

Direct interspecies electron transfer (DIET) is an effective mechanism for microbial species to exchange electrons cooperatively during syntrophic metabolism. It is generally accepted that DIET is mainly mediated by electrically conductive pili and outer surface c-type cytochromes (c-Cyts). However, as an extracellular matrix is ubiquitous and abundant on the surface of microorganisms, the effect and mechanism of exopolysaccharides on DIET are still unclear. This study constructed a co-culture of exopolysaccharides-deficient Geobacter sulfurreducens with Geobacter metallireducens to explore the role of exopolysaccharides in DIET. Results revealed that the deficiency of exopolysaccharides extended the metabolic period of the co-culture by 44.4% and changed the proportions of each species in the co-culture. The exopolysaccharides-deficient co-culture failed to form large, tight spherical aggregates and the expression of c-Cyts and pili was decreased. The addition of magnetite and granular activated carbon (GAC), respectively, might compensate for the functions of c-Cyts and pili in the first generation of co-culture, but the stimulatory effect on the metabolic stable period co-culture was fairly limited. These findings demonstrate that non-conductive exopolysaccharides are an important component of DIET aggregates and an extracellular matrix for DIET-required c-Cyts.

Maximum depth sequencing reveals an ON/OFF replication slippage switch and apparent in vivo selection for bifidobacterial pilus expression

The human gut microbiome, of which the genus Bifidobacterium is a prevalent and abundant member, is thought to sustain and enhance human health. Several surface-exposed structures, including so-called sortase-dependent pili, represent important bifidobacterial gut colonization factors. Here we show that expression of two sortase-dependent pilus clusters of the prototype Bifidobacterium breve UCC2003 depends on replication slippage at an intragenic G-tract, equivalents of which are present in various members of the Bifidobacterium genus. The nature and extent of this slippage is modulated by the host environment. Involvement of such sortase-dependent pilus clusters in microbe-host interactions, including bacterial attachment to the gut epithelial cells, has been shown previously and is corroborated here for one case. Using a Maximum Depth Sequencing strategy aimed at excluding PCR and sequencing errors introduced by DNA polymerase reagents, specific G-tract sequences in B. breve UCC2003 reveal a range of G-tract lengths whose plasticity within the population is functionally utilized. Interestingly, replication slippage is shown to be modulated under in vivo conditions in a murine model. This in vivo modulation causes an enrichment of a G-tract length which appears to allow biosynthesis of these sortase-dependent pili. This work provides the first example of productive replication slippage influenced by in vivo conditions. It highlights the potential for microdiversity generation in “beneficial” gut commensals.

The lncRNA PILA promotes NF-κB signaling in osteoarthritis by stimulating the activity of the protein arginine methyltransferase PRMT1

Inflammatory cytokine-induced activation of nuclear factor κB (NF-κB) signaling plays a critical role in the pathogenesis of osteoarthritis (OA). We identified PILA as a long noncoding RNA (lncRNA) that enhances NF-κB signaling and OA. The abundance of PILA was increased in damaged cartilage from patients with OA and in human articular chondrocytes stimulated with the proinflammatory cytokine tumor necrosis factor (TNF). Knockdown of PILA inhibited TNF-induced NF-κB signaling, extracellular matrix catabolism, and apoptosis in chondrocytes, whereas ectopic expression of PILA promoted NF-κB signaling and matrix degradation. PILA promoted PRMT1-mediated arginine methylation of DExH-box helicase 9 (DHX9), leading to an increase in the transcription of the gene encoding transforming growth factor β-activated kinase 1 (TAK1), an upstream activator of NF-κB signaling. Furthermore, intra-articular injection of an adenovirus vector encoding PILA triggered spontaneous cartilage loss and exacerbated posttraumatic OA in mice. This study provides insight into the regulation of NF-κB signaling in OA and identifies a potential therapeutic target for this disease.

Comparing Pili Producing Gene (mtp-flp) in Susceptible and Resistant Dormant Mycobacterium Tuberculosis Strains with Active Clinical Isolates

Background: Pili producing genes in different life cycles of Mycobacterium tuberculosis (M. tuberculosis) were assessed. M. tuberculosis has two life cycles: dormant and active states. We aimed to assess the pili producing genes such as curli pili of M. tuberculosis (mtp) encoded by the mtp gene (Rv3312A) and fimbrial low-molecular-weight protein encoded by flp gene (Rv3656c) which were compared and analyzed. Methods: Two hundred M. tuberculosis isolates were investigated both at active and dormant states for production and expression of pili. The dormant M. tuberculosis was achieved by incubation in a sealed tube (modified Wayne method). The susceptibility of M. tuberculosis was evaluated on genes, rpob, inh, katg, and gyra by using multiplex polymerase chain reaction (PCR) and single-strand conformational polymorphism methods. The PCR–restriction fragment length polymorphism was used to express pili genes mtp and flp and then the PCR products was digested using restriction enzyme Fnu4HI, XmaI, and MspJI and AciI, TagII, and HaeII, respectively. The transmission electron microscopy was also used to detect pili in different isolates. The result was compared and analyzed using H37RV as a gold standard. Results: The mtp and flp PCR products were 263 and 122 bp in the studied strains irrespective of M. tuberculosis different life cycles, respectively. The PCR products were analyzed on 8% Polyacrylamide gel electrophoresis (PAGE), and in the 180/200 (20%), producing five fragments of 25,40,45,63,90 bp with the Fun4HI and two fragments of 126,138 bp with the XmaI and uncut with the MspJI for mtp gen were obtained at the dormant and active states of M. tuberculosis (P < 0.05). Similarly in flp gene producing three fragments of 22,35,65 bp with AciI and two fragments of 35.87 bp with TagII and two fragments of 38.84 bp with HaeII were obtained (P < 0.05). In contrast to genotyping analysis, the electron microscopy examination showed protruding of pili from M. tuberculosis, especially in dormant mycobacterium (15/100; 15%), that was multidrug resistance and extensive drug resistance isolates (P > 0.05). Conclusion: Pili were shown by electron microscopy, although at the gene expression, the insignificant difference was observed at the dormant strains in comparison to active states. Therefore, we may conclude that other genes might be involved in pili production of M. tuberculosis that needs further investigation. Although, the resistance phenomena might influence the pili producing gene expression that showed in our results.