Viral afterlife: SARS-CoV-2 as a reservoir of immunomimetic peptides that reassemble into proinflammatory supramolecular complexes.

It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 (COVID-19), with amplified immune activation in diverse cell types, including cells without angiotensin-converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS-CoV-2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self-organization in the infected host environment. Interestingly, a machine learning analysis to SARS-CoV-2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL-37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS-CoV-2 relative to low-pathogenicity coronaviruses. Moreover, xenoAMPs from SARS-CoV-2 but not low-pathogenicity homologs assemble double-stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll-like receptor (TLR)-3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin's role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID-19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin-6 and CXCL1 levels as observed in COVID-19 patients.

Revisiting Host-Pathogen Interactions in Cystic Fibrosis Lungs in the Era of CFTR Modulators.

Cystic fibrosis transmembrane conductance regulator (CFTR) modulators, a new series of therapeutics that correct and potentiate some classes of mutations of the CFTR, have provided a great therapeutic advantage to people with cystic fibrosis (pwCF). The main hindrances of the present CFTR modulators are related to their limitations in reducing chronic lung bacterial infection and inflammation, the main causes of pulmonary tissue damage and progressive respiratory insufficiency, particularly in adults with CF. Here, the most debated issues of the pulmonary bacterial infection and inflammatory processes in pwCF are revisited. Special attention is given to the mechanisms favoring the bacterial infection of pwCF, the progressive adaptation of Pseudomonas aeruginosa and its interplay with Staphylococcus aureus, the cross-talk among bacteria, the bronchial epithelial cells and the phagocytes of the host immune defenses. The most recent findings of the effect of CFTR modulators on bacterial infection and the inflammatory process are also presented to provide critical hints towards the identification of relevant therapeutic targets to overcome the respiratory pathology of pwCF.

Cystic Fibrosis Airway Mucus Hyperconcentration Produces a Vicious Cycle of Mucin, Pathogen, and Inflammatory Interactions that Promotes Disease Persistence.

The dynamics describing the vicious cycle characteristic of cystic fibrosis (CF) lung disease, initiated by stagnant mucus and perpetuated by infection and inflammation, remain unclear. Here we determine the effect of the CF airway milieu, with persistent mucoobstruction, resident pathogens, and inflammation, on the mucin quantity and quality that govern lung disease pathogenesis and progression. The concentrations of MUC5AC and MUC5B were measured and characterized in sputum samples from subjects with CF (N = 44) and healthy subjects (N = 29) with respect to their macromolecular properties, degree of proteolysis, and glycomics diversity. These parameters were related to quantitative microbiome and clinical data. MUC5AC and MUC5B concentrations were elevated, 30- and 8-fold, respectively, in CF as compared with control sputum. Mucin parameters did not correlate with hypertonic saline, inhaled corticosteroids, or antibiotics use. No differences in mucin parameters were detected at baseline versus during exacerbations. Mucin concentrations significantly correlated with the age and sputum human neutrophil elastase activity. Although significantly more proteolytic cleavages were detected in CF mucins, their macromolecular properties (e.g., size and molecular weight) were not significantly different than control mucins, likely reflecting the role of S-S bonds in maintaining multimeric structures. No evidence of giant mucin macromolecule reflecting oxidative stress-induced cross-linking was found. Mucin glycomic analysis revealed significantly more sialylated glycans in CF, and the total abundance of nonsulfated O-glycans correlated with the relative abundance of pathogens. Collectively, the interaction of mucins, pathogens, epithelium, and inflammatory cells promotes proteomic and glycomic changes that reflect a persistent mucoobstructive, infectious, and inflammatory state.

Cautionary Notes on the Use of Arabinose- and Rhamnose-Inducible Expression Vectors in Pseudomonas aeruginosa.

The Pseudomonas aeruginosa virulence factor regulator (Vfr) is a cyclic AMP (cAMP)-responsive transcription factor homologous to the Escherichia coli cAMP receptor protein (CRP). Unlike CRP, which plays a central role in E. coli energy metabolism and catabolite repression, Vfr is primarily involved in the control of P. aeruginosa virulence factor expression. Expression of the Vfr regulon is controlled at the level of vfr transcription, Vfr translation, cAMP synthesis, and cAMP degradation. While investigating mechanisms that regulate Vfr translation, we placed vfr transcription under the control of the rhaBp rhamnose-inducible promoter system (designated PRha) and found that PRha promoter activity was highly dependent upon vfr. Vfr dependence was also observed for the araBp arabinose-inducible promoter (designated PBAD). The observation of Vfr dependence was not entirely unexpected. Both promoters are derived from E. coli, where maximal promoter activity is dependent upon CRP. Like CRP, we found that Vfr directly binds to promoter probes derived from the PRha and PBAD promoters in vitro. Because Vfr-cAMP activity is highly integrated into numerous global regulatory systems, including c-di-GMP signaling, the Gac/Rsm system, MucA/AlgU/AlgZR signaling, and Hfq/sRNAs, the potential exists for significant variability in PRha and PBAD promoter activity in a variety of genetic backgrounds, and use of these promoter systems in P. aeruginosa should be employed with caution. IMPORTANCE Heterologous gene expression and complementation constitute a valuable and widely utilized tool in bacterial genetics. The arabinose-inducible ParaBAD (PBAD) and rhamnose-inducible PrhaBAD (PRha) promoter systems are commonly used in P. aeruginosa genetics and prized for the tight control and dynamic expression ranges that can be achieved. In this study, we demonstrate that the activity of both promoters is dependent upon the cAMP-dependent transcription factor Vfr. While this poses an obvious problem for use in a vfr mutant background, the issue is more pervasive, considering that vfr transcription/synthesis and cAMP homeostasis are highly integrated into the cellular physiology of the organism and influenced by numerous global regulatory systems. Fortunately, the synthetic PTac promoter is not subject to Vfr regulatory control.

Evaluation of e-liquid toxicity using an open-source high-throughput screening assay.

The e-liquids used in electronic cigarettes (E-cigs) consist of propylene glycol (PG), vegetable glycerin (VG), nicotine, and chemical additives for flavoring. There are currently over 7,700 e-liquid flavors available, and while some have been tested for toxicity in the laboratory, most have not. Here, we developed a 3-phase, 384-well, plate-based, high-throughput screening (HTS) assay to rapidly triage and validate the toxicity of multiple e-liquids. Our data demonstrated that the PG/VG vehicle adversely affected cell viability and that a large number of e-liquids were more toxic than PG/VG. We also performed gas chromatography-mass spectrometry (GC-MS) analysis on all tested e-liquids. Subsequent nonmetric multidimensional scaling (NMDS) analysis revealed that e-liquids are an extremely heterogeneous group. Furthermore, these data indicated that (i) the more chemicals contained in an e-liquid, the more toxic it was likely to be and (ii) the presence of vanillin was associated with higher toxicity values. Further analysis of common constituents by electron ionization revealed that the concentration of cinnamaldehyde and vanillin, but not triacetin, correlated with toxicity. We have also developed a publicly available searchable website (www.eliquidinfo.org). Given the large numbers of available e-liquids, this website will serve as a resource to facilitate dissemination of this information. Our data suggest that an HTS approach to evaluate the toxicity of multiple e-liquids is feasible. Such an approach may serve as a roadmap to enable bodies such as the Food and Drug Administration (FDA) to better regulate e-liquid composition.

Initial acquisition and succession of the cystic fibrosis lung microbiome is associated with disease progression in infants and preschool children.

The cystic fibrosis (CF) lung microbiome has been studied in children and adults; however, little is known about its relationship to early disease progression. To better understand the relationship between the lung microbiome and early respiratory disease, we characterized the lower airways microbiome using bronchoalveolar lavage (BAL) samples obtained from clinically stable CF infants and preschoolers who underwent bronchoscopy and chest computed tomography (CT). Cross-sectional samples suggested a progression of the lower airways microbiome with age, beginning with relatively sterile airways in infancy. By age two, bacterial sequences typically associated with the oral cavity dominated lower airways samples in many CF subjects. The presence of an oral-like lower airways microbiome correlated with a significant increase in bacterial density and inflammation. These early changes occurred in many patients, despite the use of antibiotic prophylaxis in our cohort during the first two years of life. The majority of CF subjects older than four harbored a pathogen dominated airway microbiome, which was associated with a further increase in inflammation and the onset of structural lung disease, despite a negligible increase in bacterial density compared to younger patients with an oral-like airway microbiome. Our findings suggest that changes within the CF lower airways microbiome occur during the first years of life and that distinct microbial signatures are associated with the progression of early CF lung disease.

Pseudomonas aeruginosa exoproducts determine antibiotic efficacy against Staphylococcus aureus.

Chronic coinfections of Staphylococcus aureus and Pseudomonas aeruginosa frequently fail to respond to antibiotic treatment, leading to significant patient morbidity and mortality. Currently, the impact of interspecies interaction on S. aureus antibiotic susceptibility remains poorly understood. In this study, we utilize a panel of P. aeruginosa burn wound and cystic fibrosis (CF) lung isolates to demonstrate that P. aeruginosa alters S. aureus susceptibility to bactericidal antibiotics in a variable, strain-dependent manner and further identify 3 independent interactions responsible for antagonizing or potentiating antibiotic activity against S. aureus. We find that P. aeruginosa LasA endopeptidase potentiates lysis of S. aureus by vancomycin, rhamnolipids facilitate proton-motive force-independent tobramycin uptake, and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) induces multidrug tolerance in S. aureus through respiratory inhibition and reduction of cellular ATP. We find that the production of each of these factors varies between clinical isolates and corresponds to the capacity of each isolate to alter S. aureus antibiotic susceptibility. Furthermore, we demonstrate that vancomycin treatment of a S. aureus mouse burn infection is potentiated by the presence of a LasA-producing P. aeruginosa population. These findings demonstrate that antibiotic susceptibility is complex and dependent not only upon the genotype of the pathogen being targeted, but also on interactions with other microorganisms in the infection environment. Consideration of these interactions will improve the treatment of polymicrobial infections.

RsmV, a Small Noncoding Regulatory RNA in Pseudomonas aeruginosa That Sequesters RsmA and RsmF from Target mRNAs.

The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has distinct genetic programs that favor either acute or chronic virulence gene expression. Acute virulence is associated with twitching and swimming motility, expression of a type III secretion system (T3SS), and the absence of alginate, Psl, or Pel polysaccharide production. Traits associated with chronic infection include growth as a biofilm, reduced motility, and expression of a type VI secretion system (T6SS). The Rsm posttranscriptional regulatory system plays important roles in the inverse control of phenotypes associated with acute and chronic virulence. RsmA and RsmF are RNA-binding proteins that interact with target mRNAs to control gene expression at the posttranscriptional level. Previous work found that RsmA activity is controlled by at least three small, noncoding regulatory RNAs (RsmW, RsmY, and RsmZ). In this study, we took an in silico approach to identify additional small RNAs (sRNAs) that might function in the sequestration of RsmA and/or RsmF (RsmA/RsmF) and identified RsmV, a 192-nucleotide (nt) transcript with four predicted RsmA/RsmF consensus binding sites. RsmV is capable of sequestering RsmA and RsmF in vivo to activate translation of tssA1, a component of the T6SS, and to inhibit T3SS gene expression. Each of the predicted RsmA/RsmF consensus binding sites contributes to RsmV activity. Electrophoretic mobility shifts assays show that RsmF binds RsmV with >10-fold higher affinity than RsmY and RsmZ. Gene expression studies revealed that the temporal expression pattern of RsmV differs from those of RsmW, RsmY, and RsmZ. These findings suggest that each sRNA may play a distinct role in controlling RsmA and RsmF activity.IMPORTANCE The members of the CsrA/RsmA family of RNA-binding proteins play important roles in posttranscriptional control of gene expression. The activity of CsrA/RsmA proteins is controlled by small noncoding RNAs that function as decoys to sequester CsrA/RsmA from target mRNAs. Pseudomonas aeruginosa has two CsrA family proteins (RsmA and RsmF) and at least four sequestering sRNAs (RsmV [identified in this study], RsmW, RsmY, and RsmZ) that control RsmA/RsmF activity. RsmY and RsmZ are the primary sRNAs that sequester RsmA/RsmF, and RsmV and RsmW appear to play smaller roles. Differences in the temporal and absolute expression levels of the sRNAs and in their binding affinities for RsmA/RsmF may provide a mechanism of fine-tuning the output of the Rsm system in response to environmental cues.

Functional Analyses of the RsmY and RsmZ Small Noncoding Regulatory RNAs in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA.IMPORTANCE CsrA homologs are present in many bacteria. The opportunistic pathogen Pseudomonas aeruginosa uses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activity in vivo Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activity in vivo and in vitro are more stringent than those for RsmA.

Anaerobic bacteria cultured from cystic fibrosis airways correlate to milder disease: a multisite study.

Anaerobic and aerobic bacteria were quantitated in respiratory samples across three cystic fibrosis (CF) centres using extended culture methods. Subjects aged 1-69 years who were clinically stable provided sputum (n=200) or bronchoalveolar lavage (n=55). 18 anaerobic and 39 aerobic genera were cultured from 59% and 95% of samples, respectively; 16 out of 57 genera had a ≥5% prevalence across centres.Analyses of microbial communities using co-occurrence networks in sputum samples showed groupings of oral, including anaerobic, bacteria, whereas typical CF pathogens formed distinct entities. Pseudomonas was associated with worse nutrition and F508del genotype, whereas anaerobe prevalence was positively associated with pancreatic sufficiency, better nutrition and better lung function. A higher total anaerobe/total aerobe CFU ratio was associated with pancreatic sufficiency and better nutrition. Subjects grouped by factor analysis who had relative dominance of anaerobes over aerobes had milder disease compared with a Pseudomonas-dominated group with similar proportions of subjects that were homozygous for F508del.In summary, anaerobic bacteria occurred at an early age. In sputum-producing subjects anaerobic bacteria were associated with milder disease, suggesting that targeted eradication of anaerobes may not be warranted in sputum-producing CF subjects.

Phosphoryl Group Flow within the Pseudomonas aeruginosa Pil-Chp Chemosensory System: DIFFERENTIAL FUNCTION OF THE EIGHT PHOSPHOTRANSFERASE AND THREE RECEIVER DOMAINS.

Bacterial chemosensory signal transduction systems that regulate motility by type IV pili (T4P) can be markedly more complex than related flagellum-based chemotaxis systems. In T4P-based systems, the CheA kinase often contains numerous potential sites of phosphorylation, but the signaling mechanisms of these systems are unknown. In Pseudomonas aeruginosa, the Pil-Chp system regulates T4P-mediated twitching motility and cAMP levels, both of which play roles in pathogenesis. The Pil-Chp histidine kinase (ChpA) has eight "Xpt" domains; six are canonical histidine-containing phosphotransfer (Hpt) domains and two have a threonine (Tpt) or serine (Spt) in place of the histidine. Additionally, there are two stand-alone receiver domains (PilG and PilH) and a ChpA C-terminal receiver domain (ChpArec). Here, we demonstrate that the ChpA Xpts are functionally divided into three categories as follows: (i) those phosphorylated with ATP (Hpt4-6); (ii) those reversibly phosphorylated by ChpArec (Hpt2-6), and (iii) those with no detectable phosphorylation (Hpt1, Spt, and Tpt). There was rapid phosphotransfer from Hpt2-6 to ChpArec and from Hpt3 to PilH, whereas transfer to PilG was slower. ChpArec also had a rapid rate of autodephosphorylation. The biochemical results together with in vivo cAMP and twitching phenotypes of key ChpA phosphorylation site point mutants supported a scheme whereby ChpArec functions both as a phosphate sink and a phosphotransfer element linking Hpt4-6 to Hpt2-3. Hpt2 and Hpt3 are likely the dominant sources of phosphoryl groups for PilG and PilH, respectively. The data are synthesized in a signaling circuit that contains fundamental features of two-component phosphorelays.

Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF.

UNLABELLED: CsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level. Pseudomonas aeruginosa has a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed with tssA1, an in vivo regulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent. IMPORTANCE: The CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis. P. aeruginosa has two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution to P. aeruginosa physiology and virulence.

Vfr Directly Activates exsA Transcription To Regulate Expression of the Pseudomonas aeruginosa Type III Secretion System.

UNLABELLED: The Pseudomonas aeruginosa cyclic AMP (cAMP)-Vfr system (CVS) is a global regulator of virulence gene expression. Regulatory targets include type IV pili, secreted proteases, and the type III secretion system (T3SS). The mechanism by which CVS regulates T3SS gene expression remains undefined. Single-cell expression studies previously found that only a portion of the cells within a population express the T3SS under inducing conditions, a property known as bistability. We now report that bistability is altered in avfr mutant, wherein a substantially smaller fraction of the cells express the T3SS relative to the parental strain. Since bistability usually involves positive-feedback loops, we tested the hypothesis that virulence factor regulator (Vfr) regulates the expression of exsA ExsA is the central regulator of T3SS gene expression and autoregulates its own expression. Although exsA is the last gene of the exsCEBA polycistronic mRNA, we demonstrate that Vfr directly activates exsA transcription from a second promoter (PexsA) located immediately upstream of exsA PexsA promoter activity is entirely Vfr dependent. Direct binding of Vfr to a PexsA promoter probe was demonstrated by electrophoretic mobility shift assays, and DNase I footprinting revealed an area of protection that coincides with a putative Vfr consensus-binding site. Mutagenesis of that site disrupted Vfr binding and PexsA promoter activity. We conclude that Vfr contributes to T3SS gene expression through activation of the PexsA promoter, which is internal to the previously characterized exsCEBA operon. IMPORTANCE: Vfr is a cAMP-dependent DNA-binding protein that functions as a global regulator of virulence gene expression in Pseudomonas aeruginosa Regulation by Vfr allows for the coordinate production of related virulence functions, such as type IV pili and type III secretion, required for adherence to and intoxication of host cells, respectively. Although the molecular mechanism of Vfr regulation has been defined for many target genes, a direct link between Vfr and T3SS gene expression had not been established. In the present study, we report that Vfr directly controls exsA transcription, the master regulator of T3SS gene expression, from a newly identified promoter located immediately upstream of exsA.

An unusual CsrA family member operates in series with RsmA to amplify posttranscriptional responses in Pseudomonas aeruginosa.

Members of the CsrA family of prokaryotic mRNA-binding proteins alter the translation and/or stability of transcripts needed for numerous global physiological processes. The previously described CsrA family member in Pseudomonas aeruginosa (RsmA) plays a central role in determining infection modality by reciprocally regulating processes associated with acute (type III secretion and motility) and chronic (type VI secretion and biofilm formation) infection. Here we describe a second, structurally distinct RsmA homolog in P. aeruginosa (RsmF) that has an overlapping yet unique regulatory role. RsmF deviates from the canonical 5 ß-strand and carboxyl-terminal α-helix topology of all other CsrA proteins by having the α-helix internally positioned. Despite striking changes in topology, RsmF adopts a tertiary structure similar to other CsrA family members and binds a subset of RsmA mRNA targets, suggesting that RsmF activity is mediated through a conserved mechanism of RNA recognition. Whereas deletion of rsmF alone had little effect on RsmA-regulated processes, strains lacking both rsmA and rsmF exhibited enhanced RsmA phenotypes for markers of both type III and type VI secretion systems. In addition, simultaneous deletion of rsmA and rsmF resulted in superior biofilm formation relative to the wild-type or rsmA strains. We show that RsmF translation is derepressed in an rsmA mutant and demonstrate that RsmA specifically binds to rsmF mRNA in vitro, creating a global hierarchical regulatory cascade that operates at the posttranscriptional level.

The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System.

UNLABELLED: The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence factor important for phagocytic avoidance, disruption of host cell signaling, and host cell cytotoxicity. ExsA is the master regulator of T3SS transcription. The expression, synthesis, and activity of ExsA is tightly regulated by both intrinsic and extrinsic factors. Intrinsic regulation consists of the well-characterized ExsECDA partner-switching cascade, while extrinsic factors include global regulators that alter exsA transcription and/or translation. To identify novel extrinsic regulators of ExsA, we conducted a transposon mutagenesis screen in the absence of intrinsic control. Transposon disruptions within gene PA2840, which encodes a homolog of the Escherichia coli RNA-helicase DeaD, significantly reduced T3SS gene expression. Recent studies indicate that E. coli DeaD can promote translation by relieving inhibitory secondary structures within target mRNAs. We report here that PA2840, renamed DeaD, stimulates ExsA synthesis at the posttranscriptional level. Genetic experiments demonstrate that the activity of an exsA translational fusion is reduced in a deaD mutant. In addition, exsA expression in trans fails to restore T3SS gene expression in a deaD mutant. We hypothesized that DeaD relaxes mRNA secondary structure to promote exsA translation and found that altering the mRNA sequence of exsA or the native exsA Shine-Dalgarno sequence relieved the requirement for DeaD in vivo. Finally, we show that purified DeaD promotes ExsA synthesis using in vitro translation assays. Together, these data reveal a novel regulatory mechanism for P. aeruginosa DeaD and add to the complexity of global regulation of T3SS. IMPORTANCE: Although members of the DEAD box family of RNA helicases are appreciated for their roles in mRNA degradation and ribosome biogenesis, an additional role in gene regulation is now emerging in bacteria. By relaxing secondary structures in mRNAs, DEAD box helicases are now thought to promote translation by enhancing ribosomal recruitment. We identify here an RNA helicase that plays a critical role in promoting ExsA synthesis, the central regulator of the Pseudomonas aeruginosa type III secretion system, and provide additional evidence that DEAD box helicases directly stimulate translation of target genes. The finding that DeaD stimulates exsA translation adds to a growing list of transcriptional and posttranscriptional regulatory mechanisms that control type III gene expression.

The AlgZR two-component system recalibrates the RsmAYZ posttranscriptional regulatory system to inhibit expression of the Pseudomonas aeruginosa type III secretion system.

Pseudomonas aeruginosa causes chronic airway infections in cystic fibrosis (CF) patients. A classic feature of CF airway isolates is the mucoid phenotype. Mucoidy arises through mutation of the mucA anti-sigma factor and subsequent activation of the AlgU regulon. Inactivation of mucA also results in reduced expression of the Vfr transcription factor. Vfr regulates several important virulence factors, including a type III secretion system (T3SS). In the present study, we report that ExsA expression, the master regulator of T3SS gene expression, is further reduced in mucA mutants through a Vfr-independent mechanism involving the RsmAYZ regulatory system. RsmA is an RNA binding protein required for T3SS gene expression. Genetic experiments suggest that the AlgZR two-component system, part of the AlgU regulon, inhibits ExsA expression by increasing the expression of RsmY and RsmZ, two small noncoding RNAs that sequester RsmA from target mRNAs. Epistasis analyses revealed that increasing the concentration of free RsmA, through either rsmYZ deletion or increased RsmA expression, partially restored T3SS gene expression in the mucA mutant. Furthermore, increasing RsmA availability in combination with Vfr complementation fully restored T3SS expression. Recalibration of the RsmAYZ system by AlgZR, however, did not alter the expression of other selected RsmA-dependent targets. We account for this observation by showing that ExsA expression is more sensitive to changes in free RsmA than other members of the RsmA regulon. Together, these data indicate that recalibration of the RsmAYZ system partially accounts for reduced T3SS gene expression in mucA mutants.

Mechanisms of reduced susceptibility and genotypic prediction of antibiotic resistance in Prevotella isolated from cystic fibrosis (CF) and non-CF patients.

OBJECTIVES: To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cultured from patients with cystic fibrosis (CF), patients with invasive infection and healthy control subjects and to determine whether genotype can be used to predict phenotypic resistance. METHODS: The susceptibility of 157 Prevotella isolates to seven antibiotics was compared, with detection of resistance genes (cfxA-type gene, ermF and tetQ), mutations within the CfxA-type ß-lactamase and expression of efflux pumps. RESULTS: Prevotella isolates positive for a cfxA-type gene had higher MICs of amoxicillin and ceftazidime compared with isolates negative for this gene (P < 0.001). A mutation within the CfxA-type ß-lactamase (Y239D) was associated with ceftazidime resistance (P = 0.011). The UK CF isolates were 5.3-fold, 2.7-fold and 5.7-fold more likely to harbour ermF compared with the US CF, UK invasive and UK healthy control isolates, respectively. Higher concentrations of azithromycin (P < 0.001) and clindamycin (P < 0.001) were also required to inhibit the growth of the ermF-positive isolates compared with ermF-negative isolates. Furthermore, tetQ-positive Prevotella isolates had higher MICs of tetracycline (P = 0.001) and doxycycline (P < 0.001) compared with tetQ-negative isolates. Prevotella spp. were also shown, for the first time, to express resistance nodulation division (RND)-type efflux pumps. CONCLUSIONS: This study has demonstrated that Prevotella isolated from various sources harbour a common pool of resistance genes and possess RND-type efflux pumps, which may contribute to tetracycline resistance. The findings indicate that antibiotic resistance is common in Prevotella spp., but the genotypic traits investigated do not reflect phenotypic antibiotic resistance in every instance.

Lung microbiota and bacterial abundance in patients with bronchiectasis when clinically stable and during exacerbation.

RATIONALE: Characterization of bacterial populations in infectious respiratory diseases will provide improved understanding of the relationship between the lung microbiota, disease pathogenesis, and treatment outcomes. OBJECTIVES: To comprehensively define lung microbiota composition during stable disease and exacerbation in patients with bronchiectasis. METHODS: Sputum was collected from patients when clinically stable and before and after completion of antibiotic treatment of exacerbations. Bacterial abundance and community composition were analyzed using anaerobic culture and 16S rDNA pyrosequencing. MEASUREMENTS AND MAIN RESULTS: In clinically stable patients, aerobic and anaerobic bacteria were detected in 40 of 40 (100%) and 33 of 40 (83%) sputum samples, respectively. The dominant organisms cultured were Pseudomonas aeruginosa (n = 10 patients), Haemophilus influenzae (n = 12), Prevotella (n = 18), and Veillonella (n = 13). Pyrosequencing generated more than 150,000 sequences, representing 113 distinct microbial taxa; the majority of observed community richness resulted from taxa present in low abundance with similar patterns of phyla distribution in clinically stable patients and patients at the onset of exacerbation. After treatment of exacerbation, there was no change in total (P = 0.925), aerobic (P = 0.917), or anaerobic (P = 0.683) load and only a limited shift in community composition. Agreement for detection of bacteria by culture and pyrosequencing was good for aerobic bacteria such as P. aeruginosa (κ = 0.84) but poorer for other genera including anaerobes. Lack of agreement was largely due to bacteria being detected by pyrosequencing but not by culture. CONCLUSIONS: A complex microbiota is present in the lungs of patients with bronchiectasis and remains stable through treatment of exacerbations, suggesting that changes in microbiota composition do not account for exacerbations.

Saliva sampling method influences oral microbiome composition and taxa distribution associated with oral diseases.

Saliva is a readily accessible and inexpensive biological specimen that enables investigation of the oral microbiome, which can serve as a biomarker of oral and systemic health. There are two routine approaches to collect saliva, stimulated and unstimulated; however, there is no consensus on how sampling method influences oral microbiome metrics. In this study, we analyzed paired saliva samples (unstimulated and stimulated) from 88 individuals, aged 7-18 years. Using 16S rRNA gene sequencing, we investigated the differences in bacterial microbiome composition between sample types and determined how sampling method affects the distribution of taxa associated with untreated dental caries and gingivitis. Our analyses indicated significant differences in microbiome composition between the sample types. Both sampling methods were able to detect significant differences in microbiome composition between healthy subjects and subjects with untreated caries. However, only stimulated saliva revealed a significant association between microbiome diversity and composition in individuals with diagnosed gingivitis. Furthermore, taxa previously associated with dental caries and gingivitis were preferentially enriched in individuals with each respective disease only in stimulated saliva. Our study suggests that stimulated saliva provides a more nuanced readout of microbiome composition and taxa distribution associated with untreated dental caries and gingivitis compared to unstimulated saliva.

Mucus polymer concentration and in vivo adaptation converge to define the antibiotic response of Pseudomonas aeruginosa during chronic lung infection.

The airway milieu of individuals with muco-obstructive airway diseases (MADs) is defined by the accumulation of dehydrated mucus due to hyperabsorption of airway surface liquid and defective mucociliary clearance. Pathological mucus becomes progressively more viscous with age and disease severity due to the concentration and overproduction of mucin and accumulation of host-derived extracellular DNA (eDNA). Respiratory mucus of MADs provides a niche for recurrent and persistent colonization by respiratory pathogens, including Pseudomonas aeruginosa, which is responsible for the majority of morbidity and mortality in MADs. Despite high concentration inhaled antibiotic therapies and the absence of antibiotic resistance, antipseudomonal treatment failure in MADs remains a significant clinical challenge. Understanding the drivers of antibiotic tolerance is essential for developing more effective treatments that eradicate persistent infections. The complex and dynamic environment of diseased airways makes it difficult to model antibiotic efficacy in vitro. We aimed to understand how mucin and eDNA concentrations, the two dominant polymers in respiratory mucus, alter the antibiotic tolerance of P. aeruginosa. Our results demonstrate that polymer concentration and molecular weight affect P. aeruginosa survival post antibiotic challenge. Polymer-driven antibiotic tolerance was not explicitly associated with reduced antibiotic diffusion. Lastly, we established a robust and standardized in vitro model for recapitulating the ex vivo antibiotic tolerance of P. aeruginosa observed in expectorated sputum across age, underlying MAD etiology, and disease severity, which revealed the inherent variability in intrinsic antibiotic tolerance of host-evolved P. aeruginosa populations. IMPORTANCE: Antibiotic treatment failure in Pseudomonas aeruginosa chronic lung infections is associated with increased morbidity and mortality, illustrating the clinical challenge of bacterial infection control. Understanding the underlying infection environment, as well as the host and bacterial factors driving antibiotic tolerance and the ability to accurately recapitulate these factors in vitro, is crucial for improving antibiotic treatment outcomes. Here, we demonstrate that increasing concentration and molecular weight of mucin and host eDNA drive increased antibiotic tolerance to tobramycin. Through systematic testing and modeling, we identified a biologically relevant in vitro condition that recapitulates antibiotic tolerance observed in ex vivo treated sputum. Ultimately, this study revealed a dominant effect of in vivo evolved bacterial populations in defining inter-subject ex vivo antibiotic tolerance and establishes a robust and translatable in vitro model for therapeutic development.