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Biology subjects

Rice, S. A.

Publications and source records attributed to Rice, S. A..

18 recordsLinked to original sources

Contact- and diffusion-based allelopathic interaction of a seaweed with coral holobionts

Shifts from coral-dominated to macroalgal-dominated reef systems have become increasingly common in many coastal regions worldwide. Coral-macroalgal interactions have been shown to be generally detrimental to coral health, with macroalgal allelopathic compounds able to exert serious and even lethal effects on coral at various stages of growth and development. Previous studies have shown that the coral-associated microbial communities play important roles in coral health and mitigating external environmental stress, including macroalgal contact stress. However, it remains unclear whether changes in the coral microbiome have an influence on the Symbiodiniaceae community composition, and if such changes subsequently affect coral health. In this study, we examined changes in both the coral microbiome and the Symbiodiniaceae communities of two Singaporean coral species (Pocillopora acuta and Merulina ampliata) when exposed to both direct and water-mediated macroalgal contact with Lobophora sp. This was investigated using 16S rRNA gene amplicon sequencing to characterize the coral microbiome, and ITS2 variable region sequencing to profile the Symbiodiniaceae communities. Although no significant differences were observed for the coral microbiomes and Symbiodiniaceae communities at both alpha-and beta- diversity levels between control and macroalgal contact treated fragments within each coral species, inter-colony variations in responses to macroalgal contact were observed for both the coral microbiome and Symbiodiniaceae communities of M. ampliata. These results suggest that coral colonies vary in the mechanisms that allow mitigation of the effects of macroalgal contact, and in their resilience to macroalgal-induced stress.

ecology↗

Mixed-species interactions constrain diversification and shape biofilm evolution

Long-term experimental evolution (LTEE) provides a powerful framework for dissecting how ecological interactions shape adaptive trajectories. Here, we evolved Klebsiella pneumoniae, Pseudomonas protegens and Pseudomonas aeruginosa in single- and mixed-species biofilm communities for 24 weeks and tracked changes in population dynamics, phenotypes, and genomes. In mono-species evolution, all three species exhibited similar dynamics of adaptation, with steadily increasing biofilm-associated populations. In contrast, mixed-species communities displayed striking compositional shifts, with P. protegens emerging as the dominant biofilm former and K. pneumoniae dominating the supernatant. Phenotypic assays revealed that all three species showed enhanced biofilm formation, but this increase was consistently greater in isolates from mono-species than mixed species communities, with P. protegens showing the largest gains. Beyond biofilm production, biofilm-associated isolates exhibited greater phenotypic diversification than planktonic isolates, whereas mixed-species interactions constrained diversification. Whole-genome sequencing identified species-specific putative adaptations such as csrD in K. pneumoniae, yfiBNR in P. protegens, and cheA in P. aeruginosa that arose early, persisted, and were enriched in mixed-species isolates. Functional assays confirmed that these mutations were indeed adaptive by enhancing biofilm formation, with yfiBNR mutations in P. protegens increasing cyclic-di-GMP production and producing a competitive advantage that recapitulated its dominance in LTEE biofilms. Our findings show that biofilm evolution fosters phenotypic diversification, whereas interspecific interactions shape adaptive trajectories, with specific mutations acting as keystone drivers of long-term ecological dynamics in multi-species communities.

microbiology↗

Prediction of symptomatic and asymptomatic bacteriuria in spinal cord injury patients using machine learning

BackgroundIndividuals with spinal cord injuries (SCI) frequently rely on urinary catheters to drain urine from the bladder, making them susceptible to asymptomatic and symptomatic catheter-associated bacteriuria and urinary tract infections (UTI). Proper identification of these conditions lacks precision, leading to inappropriate antibiotic use which promotes selection for drug-resistant bacteria. Since infection often leads to dysbiosis in the microbiome and correlates with health status, this study aimed to develop a machine learning-based diagnostic framework to predict potential UTI by monitoring urine and/or catheter microbiome data, thereby minimising unnecessary antibiotic use and improving patient health. ResultsMicrobial communities in 609 samples (309 catheter and 300 urine) with asymptomatic and symptomatic bacteriuria status were analysed using 16S rRNA gene sequencing from 27 participants over 18 months. Microbial community compositions were significantly different between asymptomatic and symptomatic bacteriuria, suggesting microbial community signatures have potential application as a diagnostic tool. A significant decrease in local (alpha) diversity was noted in symptomatic bacteriuria compared to the asymptomatic bacteriuria (P < 0.01). Beta diversity measured in weighted unifrac also showed a significant difference (P < 0.05) between groups. Supervised machine learning models trained on amplicon sequence variant (ASVs) counts and bacterial taxonomic abundances (Taxa) to classify symptomatic and asymptomatic bacteriuria with a 10-fold cross-validation approach. Combining urine and catheter microbiome data improved the model performance during cross-validation, yielding a mean area under the receiver operating characteristic curve (AUROC) of 0.91-0.98 (Interquartile range, IQR 0.93-0.96) and 0.78-0.91 (IQR 0.86-0.88) for ASVs and taxonomic features, respectively. ASVs and taxa features achieve a mean AUROC of 0.85-1 (IQR 0.93-0.98) and 0.69-0.99 (IQR 0.78-0.88) in the independent held-out test set, respectively, signifying their potential in differentiating symptomatic and asymptomatic bacteriuria states. ConclusionsOur findings demonstrate that signatures within catheter and urine microbiota could serve as tools to monitor the health status of SCI patients. Establishing an early warning system based on these microbial signatures could equip physicians with alternative management strategies, potentially reducing UTI episodes and associated hospital costs, thus significantly improving patient quality of life while mitigating the impact of drug-resistant UTI.

microbiology↗

Antimicrobial mechanism of in-situ plasma activated water treatment of pathogenic Escherichia coli and Staphylococcus aureus biofilms

AimsThis study investigated the efficacy and mechanisms of inactivation of against Escherichia coli UTI89 and Staphylococcus aureus NCTC8325 through an in-situ plasma-activated water (PAW) treatment. Methods and ResultsPAW was prepared by discharging atmospheric pressure cold plasma beneath the surface of sterile distilled water. The study investigated the inactivation of biofilm cells and biofilm matrix. A complete killing of biofilm cells was achieved on both of E. coli (6.76 {+/-} 0.01 log CFU/mL) and S. aureus (6.82 {+/-} 0.02 log CFU/mL). This process happened earlier in S. aureus. Simultaneously, PAW treatment disrupted the biofilm structure, inducing a significant reduction in general biofilm biomass and extracellular polymer substances (EPS) matrix. With the disruption of EPS, PAW was enabled to further interact with the bacterial membrane, causing a significant increase in membrane permeability and disrupted membrane structure. Finally, PAW treatment led to a significant accumulation of intracellular reactive oxygen and nitrogen species within the biofilm cells. ConclusionsCollectively, these findings indicate that PAW effectively inactivates biofilms by mechanically targeting the biofilm EPS matrix and biofilm cells in both gram-negative and gram-positive bacteria. Impact statementThis study contributes novel insights into plasma-activated waters mechanisms of action, particularly its impact on the biofilm extracellular polymeric substances matrix (exopolysaccharides, extracellular DNA, and protein), cell membrane permeability, depolarization, and intracellular ROS and RNS accumulation in both of Gram-positive and Gram-negative species. These findings highlight PAW-based treatments against biofilm-related challenges in antimicrobial development and water system decontamination.

microbiology↗

Genome scale metabolic modelling of human gut microbes to inform rational community design

The human gut microbiome impacts host health through metabolite production, notably short-chain fatty acids (SCFAs) derived from digestion-resistant carbohydrates (DRCs). While DRC supplementation offers a means to modulate the microbiome therapeutically, its effectiveness is often limited by the microbial communitys complexity and individual variability in microbiome functionality. We utilized genome-scale metabolic models (GEMs) from the AGORA collection to provide a system-level overview of the metabolic capabilities of human gut microbes in terms of carbohydrate trophic networks and propose improved therapeutic interventions, based on microbial community design. Our study inferred the capability of AGORA strains to consume carbohydrates of varying structural complexities--including DRCs--and to produce metabolites amenable to cross-feeding, such as SCFAs. The resulting functional database indicated that DRC-degrading abilities are rare among gut microbes, suggesting that the presence or absence of specific taxa can determine the success of DRC-based interventions. Additionally, we found that metabolite production profiles exceed family-level variation, highlighting the limitations in predicting intervention outcomes based on gut microbial composition assessed at higher taxonomic levels. In response to these findings, we integrate reverse ecology principles, network analysis and GEM community modelling to guide the design of minimal yet resilient microbial communities to better guarantee intervention response (purpose-based communities). As a proof of principle, we predicted a purpose-based community designed to enhance butyrate production when used in conjunction with DRC supplementation, that displays resilience under nutritional stress, such as amino acid restriction. We further seeded the identified purpose-based community into modelled human microbiomes previously demonstrated to accurately predict SCFA production profiles. The analysis confirmed that such intervention significantly promotes butyrate production across samples, with those that presented a comparatively lower butyrate production pre-intervention displaying the largest increase in butyrate production after seeding. Our work highlights the potential of combining GEMs with community design to infer effective microbiome interventions, ultimately leading to improved health outcomes.

ecology↗

Transcriptional Signatures Associated with the Survival of Escherichia coli Biofilm During Treatment with Plasma-Activated Water

Biofilm formation on surfaces, tools and equipment can damage their quality and lead to high repair or replacement costs. Plasma-activated water (PAW), a new technology, has shown promise in killing biofilm and non-biofilm bacteria due to its mix of reactive oxygen and nitrogen species (RONS), and in particular superoxide. However, the specific genetic mechanisms behind PAWs effectiveness, especially against biofilms, are not yet fully understood. Here, we examined the stress responses of Escherichia coli biofilms when exposed to sub-lethal PAW treatment with and without superoxide (by adding the scavenger Tiron to remove it). A 40% variation in gene expression was observed for PAW treated biofilms when compared to PAW-Tiron and controls. Specifically, PAW treatment resulted in 478 upregulated genes (> 1.5 log2FC) and 186 downregulated genes (< -1.5 log2FC) compared to the control. Pathway enrichment and biological process enrichment analysis revealed significant upregulation of sulfur metabolism, ATP-binding cassette transporter genes, amino acid metabolic/biosynthesis pathways, hypochlorite response systems and oxidative phosphorylation for biofilms treated with PAW compared to control. Knockout mutants of significantly upregulated genes associated with these pathways trxC (4.23-fold), cysP (1.58-fold) and nuoM (1.74-fold) were compared to the wild-type (WT) for their biofilm viability and intracellular RONS accumulation. Relative to PAW-treated WT,{Delta} trxC and{Delta} nuoM knockout mutants displayed significantly reduced biofilm viability (P [&le;] 0.05) confirming their role in PAW-mediated response. Interestingly,{Delta} trxC biofilms had the highest intracellular ROS accumulation, as revealed by DCFDA staining after PAW treatment. This study gives a detailed insight into how E. coli biofilms respond to oxidative stress induced by PAW. It highlights the significance of superoxide in PAWs bactericidal effects. Overall, our findings shed light on the specific genes and pathways that help E. coli biofilms survive and respond to PAW treatment, offering a new understanding of plasma technology and its anti-biofilm mechanisms.

microbiology↗

Plasma Activated Water as a Pre-Treatment Strategy in the Context of Biofilm-Infected Chronic Wounds

Healing and treatment of chronic wounds are often complicated due to biofilm formation by pathogens. Here, the efficacy of Plasma Activated Water (PAW) as a pre-treatment strategy has been investigated prior to the application of topical antiseptics polyhexamethylene biguanide, povidone iodine, and MediHoney, which are routinely used to treat chronic wounds. The efficacy of this treatment strategy was determined against biofilms of Escherichia coli formed on a plastic substratum and on a human keratinocyte monolayer substratum used as an in vitro biofilm-skin epithelial cell model. PAW pre-treatment greatly increased the killing efficacy of all the three antiseptics to eradicate the E. coli biofilms formed on the plastic and keratinocyte substrates. However, the efficacy of the combined PAW-antiseptic treatment and single treatments using PAW or antiseptic alone was lower for biofilms formed in the in vitro biofilm-skin epithelial cell model compared to the plastic substratum. Scavenging assays demonstrated that reactive species present within the PAW were largely responsible for its anti-biofilm activity. PAW treatment resulted in significant intracellular RONS accumulation within the E. coli biofilms, while also rapidly acting on the microbial membrane leading to outer membrane permeabilisation and depolarisation. Together, these factors contribute to significant cell death, potentiating the antibacterial effect of the assessed antiseptics.

microbiology↗

Transcriptomic and metabolomic characterization of antibacterial activity of Melastoma dodecandrum

Antibacterial resistance poses a significant global threat, necessitating the discovery of new therapeutic agents. Plants are a valuable source of secondary metabolites with demonstrated anticancer and antibacterial properties. In this study, we reveal that Melastoma dodecandrum exhibits both bacteriostatic and bactericidal effects against Pseudomonas aeruginosa and Staphylococcus aureus. Treatment with plant extracts results in membrane damage and a reduction in Pseudomonas swimming and swarming motility. A comparative analysis of bacterial transcriptomes exposed to Melastoma extracts and four distinct antibiotics indicates that the extracts trigger similar transcriptomic responses as triclosan, a fatty acid inhibitor. Activity-guided fractionation suggests that the antibacterial activity is not attributable to hydrolyzable tannins, but to unidentified minor compounds. Additionally, we identified 104 specialized metabolic pathways and demonstrated a high level of transcriptional coordination between these biosynthetic pathways and phytohormones, highlighting potential regulatory mechanisms in plant metabolism.

plant biology↗

Microbe-derived indole tunes organ-function and links microbe metabolites to biological ageing

To investigate the underlying molecular mechanisms on how the gut microbe metabolite, indoles, regulate host organ growth and function, germ-free male mice were mono-colonized with indole-producing wildtype Escherichia coli or tryptophanase-encoding tnaA knockout mutant indole-non-producing E. coli. The indole mutant E. coli recipient mice exhibited significant multiorgan decline and growth retardation combined with catabolism and energy deficiency despite increased food intake compared to control mice. In addition, indole mutant mice displayed malfunctional intestine, enlarged caecum, reduced numbers of colonic enterochromaffin cells and reduced circulating serotonin levels, resulting in reduced gut motility, diminished digestion, and lower energy harvest. Furthermore, indole mutant mice also displayed decreased expression of Kcnj12 gene, suggesting reduced excitability of enteric neurons thus adding to intestinal dysfunctional phenotype. In conclusion, indoles are necessary to maintain adult metabolic homeostasis across multiple organs in vivo. Impairment of indole levels results in multiorgan functional decline suggesting a mechanism whereby gut microbe metabolites may regulate biological ageing and thus increase the risk for disease. Graphical summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/525337v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@182875dorg.highwire.dtl.DTLVardef@8525e9org.highwire.dtl.DTLVardef@17c24e3org.highwire.dtl.DTLVardef@159dff4_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

Machine-learning based detection of adventitious microbes in T-cell therapy cultures using long read sequencing

Assuring that cell therapy products are safe before releasing them for use in patients is critical. Currently, compendial sterility testing for bacteria and fungi can take 7-14 days. The goal of this work was to develop a rapid untargeted approach for the sensitive detection of microbial contaminants at low abundance from low volume samples during the manufacturing process of cell therapies. We developed a long-read sequencing methodology using Oxford Nanopore Technologies MinION platform with 16S and 18S amplicon sequencing to detect USP<71> organisms and other microbial species. Reads are classified metagenomically to predict the microbial species. We used an extreme gradient boosting machine learning algorithm (XGBoost) to first assess if a sample is contaminated and second, determine whether the predicted contaminant is correctly classified or misclassified. The model was used to make a final decision on the sterility status of the input sample. An optimised experimental and bioinformatics pipeline starting from spiked species through to sequenced reads allowed for the detection of microbial samples at 10 CFU / mL using metagenomic classification. Machine learning can be coupled with long read sequencing to detect and identify sample sterility status and microbial species present in T-cell cultures, including the USP<71> organisms to 10 CFU / mL. ImportanceThis research presents a novel method for rapidly and accurately detecting microbial contaminants in cell therapy products, which is essential for ensuring patient safety. Traditional testing methods are time-consuming, taking 7-14 days, while our approach can significantly reduce this time. By combining advanced long read Nanopore sequencing techniques and machine learning, we can effectively identify the presence and types of microbial contaminants at low abundance levels. This breakthrough has the potential to improve the safety and efficiency of cell therapy manufacturing, leading to better patient outcomes and a more streamlined production process.

bioinformatics↗

Acquired fluoroquinolone resistance genes in corneal isolates of Pseudomonas aeruginosa

Fluroquinolones are widely used as an empirical therapy for pseudomonal ocular infections. Based on increasing reports on acquired fluroquinolone resistance genes in clinical isolates of Pseudomonas aeruginosa, we investigated 33 strains of P. aeruginosa isolated from the cornea of microbial keratitis patients in India and Australia between 1992 and 2018 to understand the prevalence of acquired fluroquinolone resistance genes in ocular isolates and to assess whether the possession of those genes was associated with fluoroquinolone susceptibility. We obtained the whole genome sequence of 33 isolates using Illumina MiSeq platform and investigated the prevalence of two fluoroquinolone resistance genes crpP and qnrVC1. To examine the associated mobile genetic elements of qnrVC1 positive strains, we obtained long read sequences using Oxford Nanopore MinION and performed hybrid assembly to combine long reads with Illumina short sequence reads. We further assessed mutations in QRDRs and antibiotic susceptibilities to ciprofloxacin, levofloxacin and moxifloxacin to examine the association between resistance genes and phenotype. Twenty strains possessed crpP in genetic islands characterised by possession of integrative conjugative elements. The qnrVC1 gene was carried by four isolates on class I integrons and Tn3 transposons along with aminoglycoside and beta-lactam resistance genes. We did not observe any evidence of plasmids carrying fluroquinolone resistance genes. Resistance to fluroquinolones was observed in those strains which possessed crpP, qnrVC1 and that had QRDRs mutations. The presence of crpP was not a sole cause of fluroquinolone resistance.

microbiology↗

Changes to the resistome of Pseudomonas aeruginosa clone ST308 associated with corneal infection over time

ObjectivesThis study compared the resistomes of isolates of Pseudomonas aeruginosa clone ST308 from 2018 and 1997 from India. MethodsTwo ocular clonal type ST308 isolates of Pseudomonas aeruginosa (198 and 219) isolated in 2018 and five historical isolates (31, 32, 33, 35 and 37) isolated in 1997 at the LV Prasad Eye Institute in India were analysed for their susceptibilities to ciprofloxacin, levofloxacin, gentamicin, tobramycin, piperacillin, imipenem, ceftazidime and polymyxin B. DNA was extracted using the DNeasy(R) Blood and Tissue. Paired-end library was prepared using Nextera XT DNA library preparation kit. Libraries were sequenced on Illumina(R) MiSeq bench top sequencer generating 300 bp paired-end reads. Spades v3.12.0 was used for assembly, Resfinder v3.1. for acquired resistance genes and Snippy V2 for variants calling. Integron finder v1.5.1 was used to identify the integrons present in the genomes. ResultsThe recent isolate 219 was resistant to all tested antibiotics except polymyxin while isolate 198 was resistant to ciprofloxacin, levofloxacin, gentamicin and tobramycin. Among historical isolates five were resistant to gentamicin, tobramycin and ciprofloxacin, four were resistant to levofloxacin while two were resistant to polymyxin. Twenty-four acquired resistance genes were present in the 2018 isolates compared to 11 in the historical isolates. All isolates contained the following genes encoding for aminoglycoside aph(6)-Id, aph(3')-lIb, aph(3'')-Ib), beta-lactam (blaPAO), tetracycline (tet(G)), fosfomycin (fosA), chloramphenicol (catB7), sulphonamide (sul1), quaternary ammonium (qacEdelta1) and fluoroquinolone (crpP) resistance. Isolate 198 possessed aph(3')-VI, rmtD2, qnrVC1, blaOXA-488, blaPME-1, while 219 possessed aadA1, rmtB, aac(6')-Ib-cr, blaTEM-1B, blaVIM-2, mph(E), mph(A), msr(E). In the isolate 219 genes blaTEM-1b, blaVIM-2, sul1, qnrvc1, rmtB and aadA1 were carried on class 1 integron. While an incomplete class 1 integron was also found in isolate 198 which was located on the genome where gene rmtB, blaPME-1, qnrVC1 and sul1 genes were positioned. There were no notable differences in the number of single nucleotide polymorphisms, but recent isolates carried more insertions and deletions in their genes. ConclusionP. aeruginosa ocular clonal isolates have changed over time, with strains acquiring genes and having more insertions and deletions in their chromosomal genes that confirm resistance to antibiotics. HighlightsO_LIRecent clonal ocular isolates of Pseudomonas aeruginosa from India have acquired a number of resistance genes compared to historical clones C_LIO_LIConsequently, resistance to antibiotics particularly fluoroquinolones in recent clones of P. aeruginosa appears to have increased. C_LIO_LIThe acquired resistance genes found in the recent P. aeruginosa isolates were related to mobile genetic elements. C_LI

microbiology↗

Pseudomonas aeruginosa isolates co-incubated with Acanthamoeba castellanii exhibit phenotypes similar to chronic cystic fibrosis isolates

The opportunistic pathogen, Pseudomonas aeruginosa, is ubiquitous in the environment, and in humans is capable of causing acute and chronic infections. P. aeruginosa, when co-incubated with the bacterivorous amoeba, Acanthamoeba castellanii, for extended periods, produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans, was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early stage amoeba-adapted and non-adapted counterparts. Late-stage amoeba-adapted P. aeruginosa lost competitive fitness compared to non-adapted counterparts when grown in nutrient rich media. However, non-adapted P. aeruginosa were rapidly cleared by amoeba predation, whereas late-stage amoeba-adapted isolates remained in higher numbers 24 h after ingestion by amoeba. In addition, there was reduced uptake by macrophage of amoeba-adapted isolates and reduced uptake by human neutrophils as well as increased survival in the presence of neutrophils. Our findings indicate that the selection imposed by amoeba on P. aeruginosa resulted in reduced virulence over time. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types. Author SummaryPseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans and also causes chronic infections in immune compromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by the bacterial predators, protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here we examined the effect of long-term predation on the genotype and phenotypes expressed by P. aeruginosa. We show that long-term co-incubation with protozoa resulted in mutations in the bacteria that made them less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Thus, predation by protozoa and long term colonization of the human host may represent similar environments that select for similar losses in gene functions.

microbiology↗

A Conserved Mechanism of APOBEC3 Relocalization by Herpesviral Ribonucleotide Reductase Large Subunits

An integral part of the antiviral innate immune response is the APOBEC3 family of single-stranded DNA cytosine deaminases, which inhibits virus replication through deamination-dependent and -independent activities. Viruses have evolved mechanisms to counteract these enzymes such as HIV-1 Vif-mediated formation of a ubiquitin ligase to degrade virus-restrictive APOBEC3 enzymes. A new example is Epstein-Barr virus (EBV) ribonucleotide reductase (RNR)-mediated inhibition of cellular APOBEC3B (A3B). The large subunit of the viral RNR, BORF2, causes A3B relocalization from the nucleus to cytoplasmic bodies and thereby protects viral DNA during lytic replication. Here, we use co-immunoprecipitation and immunofluorescent microscopy approaches to ask whether this mechanism is shared with the closely related {gamma}-herpesvirus Kaposis sarcoma-associated herpesvirus (KSHV) and the more distantly related -herpesvirus, herpes simplex virus-1 (HSV-1). The large RNR subunit of KSHV, ORF61, co-precipitated multiple APOBEC3s including A3B and APOBEC3A (A3A). KSHV ORF61 also caused relocalization of these two enzymes to perinuclear bodies (A3B) and to oblong cytoplasmic structures (A3A). The large RNR subunit of HSV-1, ICP6, also co-precipitated A3B and A3A and was sufficient to promote the relocalization of these enzymes from nuclear to cytoplasmic compartments. HSV-1 infection caused similar relocalization phenotypes that required ICP6. However, unlike the infectivity defects previously reported for BORF2-null EBV, ICP6 mutant HSV-1 showed normal growth rates and plaque phenotypes. These results combine to indicate that both {gamma}- and -herpesviruses use a conserved RNR-dependent mechanism to relocalize A3B and A3A and, further, suggest that HSV-1 possesses at least one additional mechanism to neutralize these antiviral enzymes.\n\nImportanceThe APOBEC3 family of DNA cytosine deaminases constitutes a vital innate immune defense against a range of different viruses. A novel counter-restriction mechanism has recently been uncovered for the {gamma}-herpesvirus EBV, in which a subunit of the viral protein known to produce DNA building blocks (ribonucleotide reductase) causes A3B to relocalize from the nucleus to the cytosol. Here, we extend these observations with A3B to include a closely related {gamma}-herpesvirus, KSHV, and to a more distantly related -herpesvirus, HSV-1. These different viral ribonucleotide reductases also caused relocalization of A3A, which is 92% identical to A3B. These studies are important because they suggest a conserved mechanism of APOBEC3 evasion by large double-stranded DNA herpesviruses. Strategies to block this host-pathogen interaction may be effective for treating infections caused by these herpesviruses.

microbiology↗

SiaABCD - A threonine phosphorylation pathway that controls biofilm formation in Pseudomonas aeruginosa

The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen. Author SummaryPseudomonas aeruginosa is a Gram-negative bacterium that is feared within clinical environments due to its potential to cause life-threatening acute and chronic infections. One cornerstone of its success is the ability to form and disperse from biofilms, which are self-made, multicellular structures that protect the individual cell from the human immune system and antibiotic treatment. As such, therapies that combine a biofilm-interference strategy and the use of antimicrobial drugs represent one of the promising strategies to tackle infections of this organism. With the current study, we gain a deeper understanding of the SiaABCD mediated biofilm formation in response to clinically relevant environmental conditions. Further, our structural and biochemical characterization of the PP2C-type protein-phosphatase SiaA and the partner switch protein SiaC suggest that both represent promising novel targets for the development of future anti-biofilms drugs based on a signal interference strategy.

microbiology↗

The biofilm matrix scaffold of Pseudomonas species consists of non-canonically base paired extracellular DNA and RNA

While extracellular DNA (eDNA) is recognized as a critical biofilm matrix component, it is not understood how it contributes to biofilm function. Here we isolate eDNA from Pseudomonas biofilms using ionic liquids, and discover that its key biophysical signatures, i.e. fluid viscoelasticity, nucleic acid conformation, and temperature and pH dependencies of gel to solution transitions, are maintained. Solid-state analysis of isolated eDNA, as a proxy for eDNA structure in biofilms, revealed non-canonical Hoogsteen base pairs, triads or tetrads involving guanine and thymine or uracil. These were less abundant in chromosomal DNA and undetected as eDNA underwent gel-sol transition. Purine-rich RNA was present in the eDNA network, which potentially enables eDNA to be the main cross-linking exopolymer in the matrix through non-canonical nucleobase interactions. Our study suggests that Pseudomonas assemble extracellular DNA and RNA into a network with viscoelastic properties, which underpin their persistence and spreading, and may aid the development of more effective controls for biofilm-associated infections.

biochemistry↗

Accessory genome contributes to the virulence and resistance of the ocular isolate of Pseudomonas aeruginosa: A complete genome analysis

Bacteria can acquire an accessory genome through the horizontal transfer of genetic elements from non-parental lineages. This leads to rapid genetic evolution allowing traits such as antibiotic resistance and virulence to spread through bacterial communities. The study of complete genomes of bacterial strains helps to understand the genomic traits associated with virulence and antibiotic resistance. We aimed to investigate the complete accessory genome of an ocular isolate of P. aeruginosa. We obtained the complete genome of the ocular isolate strain PA34 of P. aeruginosa utilising genome sequence reads from Illumina and Oxford Nanopore Technology followed by PCR to close any identified gaps. In-depth genomic analysis was performed using various bioinformatics tools. The phenotypic properties of susceptibility to heavy metals and cytotoxicity were determined to confirm expression of certain traits. The complete genome of PA34 includes a chromosome of 6.8 Mbp and two plasmids of 95.4 Kbp (pMKPA34-1) and 26.8 Kbp (pMKPA34-2). PA34 had a large accessory genome of 1,213 genes and had 543 unique genes not present in other strains. These exclusive genes encoded features related to metal and antibiotic resistance, phage integrase and transposons. At least 24 GIs were predicated in the complete chromosome, of which two were integrated into novel sites. Eleven GIs carried virulence factors or replaced pathogenic genes. A bacteriophage carried the aminoglycoside resistance gene (aac(3)-IId). The two plasmids carried other six antibiotic resistance genes. The large accessory genome of this ocular isolate plays a large role in shaping its virulence and antibiotic resistance.

microbiology↗

Cyclic-di-GMP is required for corneal infection by Pseudomonas aeruginosa and modulates host immunity

Biofilms are extremely tolerant toward antimicrobial treatment and host immune clearance due to their distinct physiology and protection by extracellular polymeric substances. Bis-(3{acute}-5{acute})-cyclic dimeric guanosine monophosphate (c-di-GMP) is an essential messenger that regulates biofilm formation by a wide range of bacteria. However, there is a lack of physiological characterization of biofilms in vivo as well as the roles of c-di-GMP signaling in mediating host-biofilm interactions. Here, we employed dual RNA-Seq to characterize the host and pathogen transcriptomes during Pseudomonas aeruginosa infection using a mouse keratitis model. In vivo P. aeruginosa biofilms maintained a distinct physiology compared with in vitro P. aeruginosa biofilms, with enhanced virulence and iron uptake capacity. C-di-GMP synthesis was enhanced in P. aeruginosa cells in vivo, potentially due to down-regulation of the expression of several phosphodiesterases (e.g., DipA, NbdA). Increased intracellular c-di-GMP levels were required for long-term ocular colonization of P. aeruginosa and impaired host innate immunity.

microbiology↗