Cystic fibrosis (CF) is characterized by defective mucociliary clearance and chronic airway infection by a complex microbiota. Infection, persistent inflammation and periodic episodes of acute pulmonary exacerbation contribute to an irreversible decline in CF lung function. While the factors leading to acute exacerbations are poorly understood, antibiotic treatment can temporarily resolve pulmonary symptoms and partially restore lung function. Previous studies indicated that exacerbations may be associated with changes in microbial densities and the acquisition of new microbial species. Given the complexity of the CF microbiota, we applied massively parallel pyrosequencing to identify changes in airway microbial community structure in 23 adult CF patients during acute pulmonary exacerbation, after antibiotic treatment and during periods of stable disease. Over 350,000 sequences were generated, representing nearly 170 distinct microbial taxa. Approximately 60% of sequences obtained were from the recognized CF pathogens Pseudomonas and Burkholderia, which were detected in largely non-overlapping patient subsets. In contrast, other taxa including Prevotella, Streptococcus, Rothia and Veillonella were abundant in nearly all patient samples. Although antibiotic treatment was associated with a small decrease in species richness, there was minimal change in overall microbial community structure. Furthermore, microbial community composition was highly similar in patients during an exacerbation and when clinically stable, suggesting that exacerbations may represent intrapulmonary spread of infection rather than a change in microbial community composition. Mouthwash samples, obtained from a subset of patients, showed a nearly identical distribution of taxa as expectorated sputum, indicating that aspiration may contribute to colonization of the lower airways. Finally, we observed a strong correlation between low species richness and poor lung function. Taken together, these results indicate that the adult CF lung microbiome is largely stable through periods of exacerbation and antibiotic treatment and that short-term compositional changes in the airway microbiota do not account for CF pulmonary exacerbations.

Summary: Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signalling molecule adenosine 3′, 5′‐cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis‐like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP‐dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP‐dependent twitching motility is cAMP‐independent. Overall, our data define a novel function for a chemotaxis‐like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP‐dependent virulence systems.

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. CF lung disease is characterized by persistent airway infection by complex microbial communities. These communities often consist of pathogens and endogenous microbes typically associated with the oral cavity. The development of these complex communities and their relationship to CF lung disease progression is unclear. To understand the evolution of the CF lower airways microbiome, we applied sensitive molecular detection methods to characterize the bacterial DNA sequences in bronchoalveolar lavage (BAL) samples obtained from clinically stable infants and preschoolers who underwent bronchoscopy. Our findings demonstrate that CF infants have relatively sterile lower airways with a progressive shift to a microbiome dominated by aerobic and anaerobic bacterial species commonly associated with the oral cavity. This initial acquisition of a lower airways microbiome was associated with a significant increase in bacterial burden and increased airway inflammation. Transition from an oral dominated to a pathogen dominated lower airways microbiome correlated with a further increase in inflammation and the onset of structural disease despite a negligible increase in bacterial density. Our findings suggest that oral microbes may play an important role in early CF airway disease and could potentially predispose subjects to subsequent infection by pathogens.

Pseudomonas aeruginosa is a metabolically versatile environmental bacterium and an opportunistic human pathogen that relies on numerous signaling pathways to sense, respond, and adapt to fluctuating environmental cues. Although the environmental signals sensed by these pathways are poorly understood, they are largely responsible for determining whether P. aeruginosa adopts a planktonic or sessile lifestyle. These environmental lifestyle extremes parallel the acute and chronic infection phenotypes observed in human disease. In this review, we focus on four major pathways (cAMP/Vfr and c-di-GMP signaling, quorum sensing, and the Gac/Rsm pathway) responsible for sensing and integrating external stimuli into coherent regulatory control at the transcriptional, translational, and post-translational level. A common theme among these pathways is the inverse control of factors involved in promoting motility and acute infection and those associated with biofilm formation and chronic infection. In many instances these regulatory pathways influence one another, forming a complex network allowing P. aeruginosa to assimilate numerous external signals into an integrated regulatory circuit that controls a lifestyle continuum.

Vfr is a global regulator of virulence factor expression in the human pathogenPseudomonas aeruginosa. Although indirect evidence suggests that Vfr activity is controlled by cyclic AMP (cAMP), it has been hypothesized that the putative cAMP binding pocket of Vfr may accommodate additional cyclic nucleotides. In this study, we used two different approaches to generate apo-Vfr and examined its ability to bind a representative set of virulence gene promoters in the absence and presence of different allosteric effectors. Of the cyclic nucleotides tested, only cAMP was able to restore DNA binding activity to apo-Vfr. In contrast, cGMP was capable of inhibiting cAMP-Vfr DNA binding. Further, we demonstrate thatvfrexpression is autoregulated and cAMP dependent and involves Vfr binding to a previously unidentified site within thevfrpromoter region. Using a combination ofin vitroandin vivoapproaches, we show that cAMP is required for Vfr-dependent regulation of a specific subset of virulence genes. In contrast, we discovered that Vfr controls expression of thelasRpromoter in a cAMP-independent manner. In summary, our data support a model in which Vfr controls virulence gene expression by distinct (cAMP-dependent and -independent) mechanisms, which may allowP. aeruginosato fine-tune its virulence program in response to specific host cues or environments.

Tissue damage predisposes humans to life-threatening disseminating infection by the opportunistic pathogen Pseudomonas aeruginosa. Bacterial adherence to host tissue is a critical first step in this infection process. It is well established that P. aeruginosa attachment to host cells involves type IV pili (TFP), which are retractile surface fibres. The molecular details of attachment and the identity of the bacterial adhesin and host receptor remain controversial. Using a mucosal epithelium model system derived from primary human tissue, we show that the pilus-associated protein PilY1 is required for bacterial adherence. We establish that P. aeruginosa preferentially binds to exposed basolateral host cell surfaces, providing a mechanistic explanation for opportunistic infection of damaged tissue. Further, we demonstrate that invasion and fulminant infection of intact host tissue requires the coordinated and mutually dependent action of multiple bacterial factors, including pilus fibre retraction and the host cell intoxication system, termed type III secretion. Our findings offer new and important insights into the complex interactions between a pathogen and its human host and provide compelling evidence that PilY1 serves as the principal P. aeruginosa adhesin for human tissue and that it specifically recognizes a host receptor localized or enriched on basolateral epithelial cell surfaces.