Combining FAIMS based glycoproteomics and DIA proteomics reveals widespread proteome alterations in response to glycosylation occupancy changes in Neisseria gonorrhoeae.

Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within the Neisseria genus O-linked protein glycosylation is conserved yet closely related Neisseria species express O-oligosaccharyltransferases (PglOs) with distinct targeting activities. Within this work, we explore the targeting capacity of different PglOs using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of the impact of changes in glycosylation on the proteome of Neisseria gonorrhoeae. We demonstrate FAIMS expands the known glycoproteome of wild type N. gonorrhoeae MS11 and enables differences in glycosylation to be assessed across strains expressing different pglO allelic chimeras with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics, we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy occurs independently of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. This work thus expands our understanding of the N. gonorrhoeae glycoproteome and the roles that pglO allelic variation may play in governing genus-level protein glycosylation.

A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes.

Riboswitches are RNA elements acting in cis, controlling expression of their downstream genes through a metabolite-induced alteration of their secondary structure. Here, we demonstrate that two S-adenosylmethionine (SAM) riboswitches, SreA and SreB, can also function in trans and act as noncoding RNAs in Listeria monocytogenes. SreA and SreB control expression of the virulence regulator PrfA by binding to the 5'-untranslated region of its mRNA. Absence of the SAM riboswitches SreA and SreB increases the level of PrfA and virulence gene expression in L. monocytogenes. Thus, the impact of the SAM riboswitches on PrfA expression highlights a link between bacterial virulence and nutrient availability. Together, our results uncover an unexpected role for riboswitches and a distinct class of regulatory noncoding RNAs in bacteria.

RNA thermosensors facilitate Streptococcus pneumoniae and Haemophilus influenzae immune evasion.

Bacterial meningitis is a major cause of death and disability in children worldwide. Two human restricted respiratory pathogens, Streptococcus pneumoniae and Haemophilus influenzae, are the major causative agents of bacterial meningitis, attributing to 200,000 deaths annually. These pathogens are often part of the nasopharyngeal microflora of healthy carriers. However, what factors elicit them to disseminate and cause invasive diseases, remain unknown. Elevated temperature and fever are hallmarks of inflammation triggered by infections and can act as warning signals to pathogens. Here, we investigate whether these respiratory pathogens can sense environmental temperature to evade host complement-mediated killing. We show that productions of two vital virulence factors and vaccine components, the polysaccharide capsules and factor H binding proteins, are temperature dependent, thus influencing serum/opsonophagocytic killing of the bacteria. We identify and characterise four novel RNA thermosensors in S. pneumoniae and H. influenzae, responsible for capsular biosynthesis and production of factor H binding proteins. Our data suggest that these bacteria might have independently co-evolved thermosensing abilities with different RNA sequences but distinct secondary structures to evade the immune system.

Characterization of a transcriptional TPP riboswitch in the human pathogen Neisseria meningitidis.

Increasing evidence has demonstrated that regulatory RNA elements such as riboswitches (RS) play a pivotal role in the fine-tuning of bacterial gene expression. In this study, we investigated and characterized a novel transcriptional thiamine pyrophosphate (TPP) RS in the obligate human pathogen N. meningitidis MC58 (serogroup B). This RS is located in the 5´ untranslated region upstream of thiC gene, encoding a protein involved in TPP biosynthesis, an essential cofactor for all living beings. Primer extension revealed the transcriptional start site of thiC. Northern blot analysis of thiC mRNA and reporter gene studies confirmed the presence of an active TPP-sensing RS. Expression patterns of the wild-type RS and site-specific mutants showed that it is an OFF switch that controls transcription elongation of thiC mRNA. Interestingly, the regulatory mechanism of the meningococcal thiC RS resembles the Gram-positive Bacillus subtilis thiC RS rather than the Gram-negative Escherichia coli thiC RS. Therefore, the meningococcal thiC RS represents a rare example of transcriptional RS in a Gram-negative bacterium. We further observed that the RS is actively involved in modulating gene expression in response to different growth media and to supplemented bacterial and eukaryotic cell lysates as possible sources of nutrients in the nasopharynx. Our results suggest that RS-mediated gene regulation could influence meningococcal fitness, through the fine-tuning of biosynthesis and scavenging of nutrients and cofactors, such as thiamine.

Flame-Made Calcium Phosphate Nanoparticles with High Drug Loading for Delivery of Biologics.

Nanoparticles exhibit potential as drug carriers in biomedicine due to their high surface-to-volume ratio that allows for facile drug loading. Nanosized drug delivery systems have been proposed for the delivery of biologics facilitating their transport across epithelial layers and maintaining their stability against proteolytic degradation. Here, we capitalize on a nanomanufacturing process famous for its scalability and reproducibility, flame spray pyrolysis, and produce calcium phosphate (CaP) nanoparticles with tailored properties. The as-prepared nanoparticles are loaded with bovine serum albumin (model protein) and bradykinin (model peptide) by physisorption and the physicochemical parameters influencing their loading capacity are investigated. Furthermore, we implement the developed protocol by formulating CaP nanoparticles loaded with the LL-37 antimicrobial peptide, which is a biological drug currently involved in clinical trials. High loading values along with high reproducibility are achieved. Moreover, it is shown that CaP nanoparticles protect LL-37 from proteolysis in vitro. We also demonstrate that LL-37 retains its antimicrobial activity against Escherichia coli and Streptococcus pneumoniae when loaded on nanoparticles in vitro. Therefore, we highlight the potential of nanocarriers for optimization of the therapeutic profile of existing and emerging biological drugs.

Temperature triggers immune evasion by Neisseria meningitidis.

Neisseria meningitidis has several strategies to evade complement-mediated killing, and these contribute to its ability to cause septicaemic disease and meningitis. However, the meningococcus is primarily an obligate commensal of the human nasopharynx, and it is unclear why the bacterium has evolved exquisite mechanisms to avoid host immunity. Here we demonstrate that mechanisms of meningococcal immune evasion and resistance against complement increase in response to an increase in ambient temperature. We have identified three independent RNA thermosensors located in the 5' untranslated regions of genes necessary for capsule biosynthesis, the expression of factor H binding protein, and sialylation of lipopolysaccharide, which are essential for meningococcal resistance against immune killing. Therefore increased temperature (which occurs during inflammation) acts as a 'danger signal' for the meningococcus, enhancing its defence against human immune killing. Infection with viral pathogens, such as influenza, leads to inflammation in the nasopharynx with an increased temperature and recruitment of immune effectors. Thermoregulation of immune defence could offer an adaptive advantage to the meningococcus during co-infection with other pathogens, and promote the emergence of virulence in an otherwise commensal bacterium.

Thermoregulation of Meningococcal fHbp, an Important Virulence Factor and Vaccine Antigen, Is Mediated by Anti-ribosomal Binding Site Sequences in the Open Reading Frame.

During colonisation of the upper respiratory tract, bacteria are exposed to gradients of temperatures. Neisseria meningitidis is often present in the nasopharynx of healthy individuals, yet can occasionally cause severe disseminated disease. The meningococcus can evade the human complement system using a range of strategies that include recruitment of the negative complement regulator, factor H (CFH) via factor H binding protein (fHbp). We have shown previously that fHbp levels are influenced by the ambient temperature, with more fHbp produced at higher temperatures (i.e. at 37°C compared with 30°C). Here we further characterise the mechanisms underlying thermoregulation of fHbp, which occurs gradually over a physiologically relevant range of temperatures. We show that fHbp thermoregulation is not dependent on the promoters governing transcription of the bi- or mono-cistronic fHbp mRNA, or on meningococcal specific transcription factors. Instead, fHbp thermoregulation requires sequences located in the translated region of the mono-cistronic fHbp mRNA. Site-directed mutagenesis demonstrated that two anti-ribosomal binding sequences within the coding region of the fHbp transcript are involved in fHbp thermoregulation. Our results shed further light on mechanisms underlying the control of the production of this important virulence factor and vaccine antigen.

Structure and mechanism of a molecular rheostat, an RNA thermometer that modulates immune evasion by Neisseria meningitidis.

Neisseria meningitidis causes bacterial meningitis and septicemia. It evades the host complement system by upregulating expression of immune evasion factors in response to changes in temperature. RNA thermometers within mRNAs control expression of bacterial immune evasion factors, including CssA, in the 5'-untranslated region of the operon for capsule biosynthesis. We dissect the molecular mechanisms of thermoregulation and report the structure of the CssA thermometer. We show that the RNA thermometer acts as a rheostat, whose stability is optimized to respond in a small temperature range around 37°C as occur within the upper airways during infection. Small increases in temperature gradually open up the structure to allow progressively increased access to the ribosome binding site. Even small changes in stability induced by mutations of imperfect base pairs, as in naturally occurring polymorphisms, shift the thermometer response outside of the desired temperature range, suggesting that its activity could be modulated by pharmacological intervention.

Characterization of a novel antisense RNA in the major pilin locus of Neisseria meningitidis influencing antigenic variation.

UNLABELLED: Expression of type four pili (Tfp) is essential for virulence in Neisseria meningitidis. Pili mediate adhesion, bacterial aggregation, and DNA uptake. In N. meningitidis, the major pilin subunit is encoded by the pilE gene. In some strains, PilE is subject to phase and antigenic variation, which can alter Tfp properties and together offer a possible mechanism of immune escape. Pilin expression and antigenic variation can be modulated in response to environmental cues; however, the precise mechanisms of such regulation remain unclear. We identified a promoter in the pilE locus, 3' of the pilE coding sequence, on the antisense (AS) strand which is conserved in meningococci. We show that this promoter directs transcription of an AS RNA that is expressed during specific growth phases and in response to salt stress. Furthermore, we demonstrate that the transcript encompasses sequences complementary to the entire pilE coding sequence and 5' untranslated region. AS RNAs can regulate the gene on the sense strand by altering transcript stability or translation. However, by using Northern blotting, quantitative reverse transcription-PCR (RT-PCR), and Western blotting, we found no significant AS RNA-dependent changes in pilE transcript or protein level. Instead, our data indicate that the AS RNA influences pilin antigenic variation. This work provides further insights into the complex regulation of pilin expression and variation in pathogenic Neisseria. IMPORTANCE: Pathogenic Neisseria spp. express type four pili (Tfp) which are important for adhesion, aggregation and transformation. Some strains of N. meningitidis are able to vary the sequence of the major subunit (PilE) of the Tfp. The mechanisms underlying this variation are not fully defined, but the process requires several noncoding elements that are found adjacent to the pilE gene. In this work, we identified a cis-encoded RNA antisense to pilE in N. meningitidis. By using Northern blotting and RT-PCR analysis, we found that the RNA is expressed in stationary phase or following salt stress. Our work also indicates that this RNA does not significantly affect pilE or pilin expression levels but instead appears to modulate pilin variation.

The Listeria transcriptional landscape from saprophytism to virulence.

The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.

An unstructured 5'-coding region of the prfA mRNA is required for efficient translation.

Expression of virulence factors in the human bacterial pathogen Listeria monocytogenes is almost exclusively regulated by the transcriptional activator PrfA. The translation of prfA is controlled by a thermosensor located in the 5'-untranslated RNA (UTR), and is high at 37°C and low at temperatures <30°C. In order to develop a thermoregulated translational expression system, the 5'-UTR and different lengths of the prfA-coding sequences were placed in front of lacZ. When expressed in Escherichia coli, the ß-galactosidase expression was directly correlated to the length of the prfA-coding mRNA lying in front of lacZ. A similar effect was detected with gfp as a reporter gene in both L. monocytogenes and E. coli, emphasizing the requirement of the prfA-coding RNA for maximal expression. In vitro transcription/translation and mutational analysis suggests a role for the first 20 codons of the native prfA-mRNA for maximal expression. By toe-print and RNA-probing analysis, a flexible hairpin-loop located immediately downstream of the start-codon was shown to be important for ribosomal binding. The present work determines the importance of an unstructured part of the 5'-coding region of the prfA-mRNA for efficient translation.

An Automated Versatile Diagnostic Workflow for Infectious Disease Detection in Low-Resource Settings.

Laboratory automation effectively increases the throughput in sample analysis, reduces human errors in sample processing, as well as simplifies and accelerates the overall logistics. Automating diagnostic testing workflows in peripheral laboratories and also in near-patient settings -like hospitals, clinics and epidemic control checkpoints- is advantageous for the simultaneous processing of multiple samples to provide rapid results to patients, minimize the possibility of contamination or error during sample handling or transport, and increase efficiency. However, most automation platforms are expensive and are not easily adaptable to new protocols. Here, we address the need for a versatile, easy-to-use, rapid and reliable diagnostic testing workflow by combining open-source modular automation (Opentrons) and automation-compatible molecular biology protocols, easily adaptable to a workflow for infectious diseases diagnosis by detection on paper-based diagnostics. We demonstrated the feasibility of automation of the method with a low-cost Neisseria meningitidis diagnostic test that utilizes magnetic beads for pathogen DNA isolation, isothermal amplification, and detection on a paper-based microarray. In summary, we integrated open-source modular automation with adaptable molecular biology protocols, which was also faster and cheaper to perform in an automated than in a manual way. This enables a versatile diagnostic workflow for infectious diseases and we demonstrated this through a low-cost N. meningitidis test on paper-based microarrays.

Total Bacterial RNA Isolation and Northern Blotting Analysis.

The study of bacterial gene expression during infection provides vital information for researchers to understand bacterial pathogenesis and infection. The ability to obtain clean and undegraded RNA could be challenging and daunting and remains the most crucial experimental step prior to downstream analyses, such as Northern blotting, quantitative PCR (qPCR), and RNA-seq.This chapter describe two methods (acid guanidinium thiocyanate (TRIzol) phenol-chloroform and hot phenol) commonly used to isolate total bacterial RNA and are suitable for both Gram-positive and Gram-negative bacteria. Procedures such as RNA quantification and DNase treatment are also included to ensure amount and quality of the RNA samples. The second part of the chapter includes a method used to analyze bacterial gene expression (Northern blotting), two methods to generate radioactive probes, as well as target detection using a phosphorimager.

The emerging role of bacterial regulatory RNAs in disease.

Pathogenic bacteria have evolved to sense their surrounding environments and regulate their gene expression to evade host immune defences and cause disease. RNA-mediated gene expression offers a fast and energy efficient alternative to conventional transcription factors. A myriad of regulatory RNAs have been identified, especially in pathogenic bacteria. However, whether these RNAs partake in disease remains largely unexplored. Here, we review current knowledge of regulatory RNAs in human-adapted upper respiratory tract pathogens. We propose that bacterial regulatory RNAs could play important roles in disease. Elucidating the function of regulatory RNAs and identifying polymorphisms among disease isolates would provide valuable insight into their pathogeneses. Finally, we discuss the outstanding issues of regulatory RNAs in research and their applications as drug targets, therapeutics, and in providing diagnostic information predictive of disease prognosis.

Neisseria meningitidis IgA1-specific serine protease exhibits novel cleavage activity against IgG3.

Neisseria meningitidis (meningococcus) is a common bacterial colonizer of the human nasopharynx but can occasionally cause very severe systemic infections with rapid onset. Meningococci are able to degrade IgA encountered during colonization of mucosal membranes using their IgA1-specific serine protease. During systemic infection, specific IgG can induce complement-mediated lysis of the bacterium. However, meningococcal immune evasion mechanisms in thwarting IgG remain undescribed. In this study, we report for the first time that the meningococcal IgA1-specific serine protease is able to degrade IgG3 in addition to IgA. The IgG3 heavy chain is specifically cleaved in the lower hinge region thereby separating the antigen binding part from its effector binding part. Through molecular characterization, we demonstrate that meningococcal IgA1-specific serine protease of cleavage type 1 degrades both IgG3 and IgA, whereas cleavage type 2 only degrades IgA. Epidemiological analysis of 7581 clinical meningococcal isolates shows a significant higher proportion of cleavage type 1 among isolates from invasive cases compared to carrier cases, regardless of serogroup. Notably, serogroup W cc11 which is an increasing cause of invasive meningococcal disease globally harbors almost exclusively cleavage type 1 protease. Our study also shows an increasing prevalence of meningococcal isolates encoding IgA1P cleavage type 1 compared to cleavage type 2 during the observed decade (2010-2019). Altogether, our work describes a novel mechanism of IgG3 degradation by meningococci and its association to invasive meningococcal disease.

Novel hypercapsulation RNA thermosensor variants in Neisseria meningitidis and their association with invasive meningococcal disease: a genetic and phenotypic investigation and molecular epidemiological study.

BACKGROUND: Neisseria meningitidis is the causative agent of invasive meningococcal disease and the polysaccharide capsule is one of its major virulence factors. Biosynthesis of the meningococcal capsule is controlled by an RNA thermosensor (RNAT) in the 5'-untranslated region (5'-UTR) of the cssA gene. The function of the RNAT depends on an 8-bp tandem repeat configuration. We aimed to identify and characterise novel RNATs in meningococcal isolates responsible for regulating capsule production. METHODS: We investigated the allele igr_up_NEIS0055, containing the 5'-UTR of the cssA gene, in clinical meningococcal isolates for which whole-genome sequences are available on the Neisseria PubMLST database and that were isolated in Europe between Jan 1, 2010, and Dec 31, 2018. Eight isolates with different RNAT tandem repeat configurations were selected for genetic and phenotypic studies. The thermosensing capability of the RNAT and capsule production was tested with immunoblots. Bacterial survival by capsule protection was assessed with a human serum stress assay and capsule interference with bacterial cell adhesion was evaluated with a bacterial adhesion assay. The dataset of RNAT configurations was analysed for an association with invasive meningococcal disease, and was stratified to visualise the distribution of RNAT configurations within the meningococcal population. FINDINGS: Our search of PubMLST identified 112 alleles for the igr_up_NEIS0055 locus and 7013 N meningitidis isolates. Five novel RNAT tandem repeat configurations were identified and eight RNAT tandem repeat configurations, ranging from 1 × 8-bp up to 8 × 8-bp, were characterised. The disrupted RNATs (1 × 8-bp and 3 × 8-bp to 8 × 8-bp) confer upregulated CssA expression and increased capsule production compared with the native 2 × 8-bp configuration, resulting in a hypercapsulation phenotype. Increased capsule production was associated with higher survival rates in up to 25% human serum. The prevalence of a disrupted RNAT resulting in hypercapsulation was almost twice as high in invasive meningococcal disease isolates compared with carrier isolates. Disrupted RNATs were especially attributed to isolates of capsule group B and C, and clonal complexes 23, 32, 213, and 269. Hypercapsulation in one isolate led to lower adhesion onto pharyngeal cells compared with a similar isolate with low capsule production. INTERPRETATION: Six non-canonical RNAT tandem repeat variants (3 × 8-bp to 8 × 8-bp) were identified in the igr_up_NEIS0055 locus of N meningitidis that induce a hypercapsulation phenotype, thus providing the meningococci with better protection against host complement-mediated killing than does the native RNAT (2 × 8-bp). Further research is warranted to strengthen the association between hypercapsulation and the progression of invasive meningococcal disease, and to investigate the role of regulatory RNAs in meningococcal virulence and as potential markers for disease progression. FUNDING: Swedish Foundation for Strategic Research, Knut and Alice Wallenberg Foundation, and Swedish Research Council.

Point-of-Care Approaches for Meningitis Diagnosis in a Low-Resource Setting (Southwestern Uganda): Observational Cohort Study Protocol of the "PI-POC" Trial.

BACKGROUND: A timely differential diagnostic is essential to identify the etiology of central nervous system (CNS) infections in children, in order to facilitate targeted treatment, manage patients, and improve clinical outcome. OBJECTIVE: The Pediatric Infection-Point-of-Care (PI-POC) trial is investigating novel methods to improve and strengthen the differential diagnostics of suspected childhood CNS infections in low-income health systems such as those in Southwestern Uganda. This will be achieved by evaluating (1) a novel DNA-based diagnostic assay for CNS infections, (2) a commercially available multiplex PCR-based meningitis/encephalitis (ME) panel for clinical use in a facility-limited laboratory setting, (3) proteomics profiling of blood from children with severe CNS infection as compared to outpatient controls with fever yet not severely ill, and (4) Myxovirus resistance protein A (MxA) as a biomarker in blood for viral CNS infection. Further changes in the etiology of childhood CNS infections after the introduction of the pneumococcal conjugate vaccine against Streptococcus pneumoniae will be investigated. In addition, the carriage and invasive rate of Neisseria meningitidis will be recorded and serotyped, and the expression of its major virulence factor (polysaccharide capsule) will be investigated. METHODS: The PI-POC trial is a prospective observational study of children including newborns up to 12 years of age with clinical features of CNS infection, and age-/sex-matched outpatient controls with fever yet not severely ill. Participants are recruited at 2 Pediatric clinics in Mbarara, Uganda. Cerebrospinal fluid (for cases only), blood, and nasopharyngeal (NP) swabs (for both cases and controls) sampled at both clinics are analyzed at the Epicentre Research Laboratory through gold-standard methods for CNS infection diagnosis (microscopy, biochemistry, and culture) and a commercially available ME panel for multiplex PCR analyses of the cerebrospinal fluid. An additional blood sample from cases is collected on day 3 after admission. After initial clinical analyses in Mbarara, samples will be transported to Stockholm, Sweden for (1) validation analyses of a novel nucleic acid-based POC test, (2) biomarker research, and (3) serotyping and molecular characterization of S. pneumoniae and N. meningitidis. RESULTS: A pilot study was performed from January to April 2019. The PI-POC trial enrollment of patients begun in April 2019 and will continue until September 2020, to include up to 300 cases and controls. Preliminary results from the PI-POC study are expected by the end of 2020. CONCLUSIONS: The findings from the PI-POC study can potentially facilitate rapid etiological diagnosis of CNS infections in low-resource settings and allow for novel methods for determination of the severity of CNS infection in such environment. TRIAL REGISTRATION: ClinicalTrials.gov NCT03900091; https://clinicaltrials.gov/ct2/show/NCT03900091. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/21430.

Sex Steroids Induce Membrane Stress Responses and Virulence Properties in Pseudomonas aeruginosa.

Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains.IMPORTANCE Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection.

Development-dependent scavenging of nucleic acids in the filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus is an ubiquitous, filamentous and opportunistic pathogenic fungus which causes fatal invasive aspergillosis among immuno-compromised patients. Since therapeutic strategies are currently limited, the mortality rate of invasive aspergillosis is high and thus, alternative antifungal strategies are required. In this study, we demonstrate that during vegetative growth Aspergillus fumigatus is able to scavenge nucleic acids within its cell wall with accumulation rates of several thousand-fold, compared to the surrounding medium. To investigate, whether nucleic acids, attached to the fungal cell wall, are able to move further into the cytoplasm of fungal cells, we directly applied siRNAs, in the absence of lipo-transfection reagents, to growing A. fumigatus cells. In fact, addition of two 21-nt siRNA duplexes resulted in knock-down of their corresponding target mRNAs, odcA and pyrG, respectively. These findings indicate that RNA interference, mediated by siRNAs, can be used as a fast and efficient tool to investigate the functions of genes within filamentous fungi. In addition, siRNA-based therapies may provide novel approaches for antifungal treatment.