Ecological patterns and processes of temporal turnover within lung infection microbiota.

BACKGROUND: Chronic infection and consequent airway inflammation are the leading causes of morbidity and early mortality for people living with cystic fibrosis (CF). However, lower airway infections across a range of chronic respiratory diseases, including in CF, do not follow classical 'one microbe, one disease' concepts of infection pathogenesis. Instead, they are comprised of diverse and temporally dynamic lung infection microbiota. Consequently, temporal dynamics need to be considered when attempting to associate lung microbiota with changes in disease status. Set within an island biogeography framework, we aimed to determine the ecological patterns and processes of temporal turnover within the lung microbiota of 30 paediatric and adult CF patients prospectively sampled over a 3-year period. Moreover, we aimed to ascertain the contributions of constituent chronic and intermittent colonizers on turnover within the wider microbiota. RESULTS: The lung microbiota within individual patients was partitioned into constituent chronic and intermittent colonizing groups using the Leeds criteria and visualised with persistence-abundance relationships. This revealed bacteria chronically infecting a patient were both persistent and common through time, whereas intermittently infecting taxa were infrequent and rare; respectively representing the resident and transient portions of the wider microbiota. It also indicated that the extent of chronic colonization was far greater than could be appreciated with microbiological culture alone. Using species-time relationships to measure temporal turnover and Vellend's rationalized ecological processes demonstrated turnover in the resident chronic infecting groups was conserved and underpinned principally by the deterministic process of homogenizing dispersal. Conversely, intermittent colonizing groups, representing newly arrived immigrants and transient species, drove turnover in the wider microbiota and were predominately underpinned by the stochastic process of drift. For adult patients, homogenizing dispersal and drift were found to be significantly associated with lung function. Where a greater frequency of homogenizing dispersal was observed with worsening lung function and conversely drift increased with better lung function. CONCLUSIONS: Our work provides a novel ecological framework for understanding the temporal dynamics of polymicrobial infection in CF that has translational potential to guide and improve therapeutic targeting of lung microbiota in CF and across a range of chronic airway diseases. Video Abstract.

Effects of postage on recovery of pathogens from cystic fibrosis sputum samples.

BACKGROUND: Regular surveillance microbiology of sputum is used in cystic fibrosis (CF) to monitor for new pathogens and target treatments. A move to remote clinics has meant greater reliance on samples collected at home and posted back. The impact of delays and sample disruption caused by posting has not been systematically assessed but could have significant implications for CF microbiology. METHODS: Sputum samples collected from adult CF patients were mixed, split, and either processed immediately or posted back to laboratory. Processing involved a further split into aliquots for culture-dependant and-independent microbiology (quantitative PCR [QPCR] and microbiota sequencing). We calculated retrieval by both approaches for five typical CF pathogens: Pseudomonas aeruginosa, Burkholderia cepacia complex, Achromobacter xylosoxidans, Staphylococcus aureus and Stenotrophomonas maltophilia. RESULTS: 93 paired samples were collected from 73 CF patients. Median interval between sample posting and receipt was 5 days (range 1-10). For culture, overall concordance for posted and fresh samples was 86% across the five targeted pathogens (ranging from 57 to 100% for different organisms), with no bias towards either sample type. For QPCR, overall concordance was 62% (range 39-84%), again with no bias towards fresh or posted samples. There were no significant differences in culture or QPCR for samples with short (≤3days) versus extended (≥7days) postal delays. Posting had no significant impact on pathogen abundance nor on microbiota characteristics. CONCLUSIONS: Posted sputum samples reliably reproduced culture-based and molecular microbiology of freshly collected samples, even after prolonged delays at ambient conditions. This supports use of posted samples during remote monitoring.

Bacterial Culture Underestimates Lung Pathogen Detection and Infection Status in Cystic Fibrosis.

Microbiological surveillance of airway secretions is central to clinical care in cystic fibrosis (CF). However, the efficacy of microbiological culture, the diagnostic gold standard for pathogen detection, has been increasingly questioned. Here we compared culture with targeted quantitative PCR (QPCR) for longitudinal detection of 2 key pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. Prospectively collected respiratory samples taken from 20 pediatric and 20 adult CF patients over a period of 3-years were analyzed. Patients were eligible if considered free of chronic Pseudomonas infection within 12-months prior to start of study. QPCR revealed high levels of infection with both pathogens not apparent from culture alone. Pseudomonas and Staphylococcus were detected by culture on at least one sampling occasion in 12 and 29 of the patients, respectively. Conversely, both pathogens were detected in all 40 patients by QPCR. Classification of infection status also significantly altered in both pediatric and adult patients, where the number of patients deemed chronically infected with Pseudomonas and Staphylococcus increased from 1 to 28 and 9 to 34, respectively. Overall, Pseudomonas and Staphylococcus infection status classification changed respectively for 36 and 27 of all patients. In no cases did molecular identification lead to a patient being in a less clinically serious infection category. Pathogen detection and infection status classification significantly increased when assessed by QPCR in comparison to culture. This could have implications for clinical care of CF patients, including accuracy of infection diagnosis, relevant and timely antibiotic selection, antimicrobial resistance development, establishment of chronic infection, and cross-infection control. IMPORTANCE Chronic lung infection is the leading cause of morbidity and early mortality for people with cystic fibrosis (pwCF). Microbiological surveillance to detect lung pathogens is recommended as best practise in CF patient care. Here we studied pathogen detection in 40 pwCF over several years. We found that microbiological culture, the diagnostic gold standard, was significantly disparate to targeted culture-independent approaches for detection and determination of chronic infection status of two important pathogens in CF. Pathogen detection was significantly lower by culture and consequently infection status was also misclassified in most cases. In particular, the extent of chronic infection by both P. aeruginosa and S. aureus not realized with culture was striking. Our findings have implications for the development of infection and clinical care of pwCF. Future longitudinal studies with greater patient numbers will be needed to establish the full extent of the clinical implications indicated from this study.

Spatiotemporal dynamics of innate immune signaling via RIG-I-like receptors.

RIG-I, MDA5, and LGP2 comprise the RIG-I-like receptors (RLRs). RIG-I and MDA5 are essential pathogen recognition receptors sensing viral infections while LGP2 has been described as both RLR cofactor and negative regulator. After sensing and binding to viral RNA, including double-stranded RNA (dsRNA), RIG-I and MDA5 undergo cytosol-to-membrane relocalization to bind and signal through the MAVS adaptor protein on intracellular membranes, thus directing downstream activation of IRF3 and innate immunity. Here, we report examination of the dynamic subcellular localization of all three RLRs within the intracellular response to dsRNA and RNA virus infection. Observations from high resolution biochemical fractionation and electron microscopy, coupled with analysis of protein interactions and IRF3 activation, show that, in resting cells, microsome but not mitochondrial fractions harbor the central components to initiate innate immune signaling. LGP2 interacts with MAVS in microsomes, blocking the RIG-I/MAVS interaction. Remarkably, in response to dsRNA treatment or RNA virus infection, LGP2 is rapidly released from MAVS and redistributed to mitochondria, temporally correlating with IRF3 activation. We reveal that IRF3 activation does not take place on mitochondria but instead occurs at endoplasmic reticulum (ER)-derived membranes. Our observations suggest ER-derived membranes as key RLR signaling platforms controlled through inhibitory actions of LGP2 binding to MAVS wherein LGP2 translocation to mitochondria releases MAVS inhibition to facilitate RLR-mediated signaling of innate immunity.

Flavivirus Nonstructural Protein NS5 Dysregulates HSP90 to Broadly Inhibit JAK/STAT Signaling.

Pathogenic flaviviruses antagonize host cell Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling downstream of interferons α/ß. Here, we show that flaviviruses inhibit JAK/STAT signaling induced by a wide range of cytokines beyond interferon, including interleukins. This broad inhibition was mapped to viral nonstructural protein 5 (NS5) binding to cellular heat shock protein 90 (HSP90), resulting in reduced Janus kinase-HSP90 interaction and thus destabilization of unchaperoned JAKs (and other kinase clients) of HSP90 during infection by Zika virus, West Nile virus, and Japanese encephalitis virus. Our studies implicate viral dysregulation of HSP90 and the JAK/STAT pathway as a critical determinant of cytokine signaling control during flavivirus infection.

RNA-binding protein isoforms ZAP-S and ZAP-L have distinct antiviral and immune resolution functions.

The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host's ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response.