Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection.

Clinical studies report that viral infections promote acute or chronic bacterial infections at multiple host sites. These viral-bacterial co-infections are widely linked to more severe clinical outcomes. In experimental models in vitro and in vivo, virus-induced interferon responses can augment host susceptibility to secondary bacterial infection. Here, we used a cell-based screen to assess 389 interferon-stimulated genes (ISGs) for their ability to induce chronic Pseudomonas aeruginosa infection. We identified and validated five ISGs that were sufficient to promote bacterial infection. Furthermore, we dissected the mechanism of action of hexokinase 2 (HK2), a gene involved in the induction of aerobic glycolysis, commonly known as the Warburg effect. We report that HK2 upregulation mediates the induction of Warburg effect and secretion of L-lactate, which enhances chronic P. aeruginosa infection. These findings elucidate how the antiviral immune response renders the host susceptible to secondary bacterial infection, revealing potential strategies for viral-bacterial co-infection treatment.

Intern Experience Reflects Information Gathered During the Virtual Interview Process.

OBJECTIVE: The onset of the coronavirus 2019 (COVID-19) pandemic brought many changes to the residency application process including transitioning to a virtual interview platform, which continues today. The transition brought many concerns from general surgery applicants about their ability to obtain adequate information about a program virtually. We sought to characterize how information presented by programs during the first ever virtual interview cycle matched the experience of general surgery interns after training at a program for 1 year. DESIGN, SETTING, AND PARTICIPANTS: In May of 2022, a survey was distributed to 243 program directors who were asked to forward it to their general surgery categorical interns who matched during the 2021 virtual match cycle. Demographics, resources used to determine an impression of a program, and correlations between information presented virtually and what was subsequently experienced as an intern were collected. RESULTS: Forty-six program directors confirmed forwarding the survey to their categorical interns. A total of 102 general surgery interns completed the survey. Most interns (88.2%) agreed that their experience matched expectations based on information received through the virtual interview process and 98% of interns were satisfied with their experience at their training program. Interviews with faculty (40.0%), residents (68.0%) and the program web site (29.0%) were the top 3 resources used to create the most accurate impression of a program. Interns felt they were well informed during the virtual interview experience about support from fellow residents (84.3%), culture (73.0%), surgical volume (72.5%), and intern operative experience (71.6%). In addition, 65.7% of participants thought they were able to obtain a good understanding of the program's culture from the virtual process. However, 16.7% thought that their program unintentionally misrepresented aspects of the training program. CONCLUSIONS: The faculty and residency interviews were the most important factors in program ranking and most participants agreed that their virtual interview experience matched their expectations during their intern year. Most interns felt they were able to obtain a good understanding of the program's culture from the virtual process. In addition, a majority of interns felt well informed during the interview on aspects ranging from surgical volume, autonomy, and work hours to support from faculty and residents. If virtual interviews are to continue, residents can be satisfied that information gathered virtually will match the reality of their training. Programs should continue to make every effort to present their program realistically.

Iron bioavailability regulates Pseudomonas aeruginosa interspecies interactions through type VI secretion expression.

The cystic fibrosis (CF) respiratory tract harbors pathogenic bacteria that cause life-threatening chronic infections. Of these, Pseudomonas aeruginosa becomes increasingly dominant with age and is associated with worsening lung function and declining microbial diversity. We aimed to understand why P. aeruginosa dominates over other pathogens to cause worsening disease. Here, we show that P. aeruginosa responds to dynamic changes in iron concentration, often associated with viral infection and pulmonary exacerbations, to become more competitive via expression of the TseT toxic effector. However, this behavior can be therapeutically targeted using the iron chelator deferiprone to block TseT expression and competition. Overall, we find that iron concentration and TseT expression significantly correlate with microbial diversity in the respiratory tract of people with CF. These findings improve our understanding of how P. aeruginosa becomes increasingly dominant with age in people with CF and provide a therapeutically targetable pathway to help prevent this shift.

Low Diversity and Instability of the Sinus Microbiota over Time in Adults with Cystic Fibrosis.

Chronic rhinosinusitis (CRS) is a common, yet underreported and understudied manifestation of upper respiratory disease in people with cystic fibrosis (CF). Recently developed standard of care guidelines for the management of CF CRS suggest treatment of upper airway disease may ameliorate lower airway disease. We sought to determine whether changes to sinus microbial community diversity and specific taxa known to cause CF lung disease are associated with increased respiratory disease and inflammation. We performed 16S rRNA gene sequencing, supplemented with cytokine analyses, microscopy, and bacterial culturing, on samples from the sinuses of 27 adults with CF CRS. At each study visit, participants underwent endoscopic paranasal sinus sampling and clinical evaluation. We identified key drivers of microbial community composition and evaluated relationships between diversity and taxa with disease outcomes and inflammation. Sinus community diversity was low, and the composition was unstable, with many participants exhibiting alternating dominance between Pseudomonas aeruginosa and staphylococci over time. Despite a tendency for dominance by these two taxa, communities were highly individualized and shifted composition during exacerbation of sinus disease symptoms. Exacerbations were also associated with communities dominated by Staphylococcus spp. Reduced microbial community diversity was linked to worse sinus disease and the inflammatory status of the sinuses (including increased interleukin-1ß [IL-1ß]). Increased IL-1ß was also linked to worse sinus endoscopic appearance, and other cytokines were linked to microbial community dynamics. Our work revealed previously unknown instability of sinus microbial communities and a link between inflammation, lack of microbial community diversity, and worse sinus disease. IMPORTANCE Together with prior sinus microbiota studies of adults with CF chronic rhinosinusitis, our study underscores similarities between sinus and lower respiratory tract microbial community structures in CF. We show how community structure tracks with inflammation and several disease measures. This work strongly suggests that clinical management of CRS could be leveraged to improve overall respiratory health in CF. Our work implicates elevated IL-1ß in reduced microbiota diversity and worse sinus disease in CF CRS, suggesting applications for existing therapies targeting IL-1ß. Finally, the widespread use of highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has led to less frequent availability of spontaneous expectorated sputum for microbiological surveillance of lung infections. A better understanding of CF sinus microbiology could provide a much-needed alternative site for monitoring respiratory infection status by important CF pathogens.

Is There an Age Cutoff for Intracranial Pressure Monitoring?: A Propensity Score Matched Analysis of the National Trauma Data Bank.

BACKGROUND: Despite mostly favorable past evidence for use of intracranial pressure monitoring (ICPM), more recent data question not only the indications but also the utility of ICPM. The Fourth Edition Brain Trauma Foundation guidelines offer limited indications for ICPM. Evidence supports ICPM for reducing mortality in patients with severe traumatic brain injury (TBI) and cites decreased survival in elderly patients. METHODS: All patients ≥ 18 years of age with isolated TBI, head Abbreviated Injury Scale (AIS) ≥ 3, and a Glasgow Coma Scale (GCS) ≤ 8 between 2008 and 2014 were included from the National Trauma Data Bank. Exclusion criteria were head AIS = 6 and death within 24 hours. Patients with and without ICPM were compared using TBI-specific variables. Patients were then matched via propensity-score matching (PSM), and the odds ratio (OR) of death with ICPM was determined using logistic regression modeling for 8 different age strata. RESULTS: A total of 23,652 patients with a mean age of 56 years, median head AIS of 4, median GCS of 3, and overall mortality of 29.2% were analyzed. After PSM, ICPM was associated with death beginning at the age stratum of 56-65 years. Intracranial pressure monitoring was associated with survival beginning at the age-group 36-45 years. DISCUSSION: Based on a large propensity-matched sample of TBI patients, ICPM was not associated with improved survival for TBI patients above 55 years of age. Until level 1 evidence is available, this age threshold should be considered for further prospective study in determining indications for ICPM.

Adaptation and genomic erosion in fragmented Pseudomonas aeruginosa populations in the sinuses of people with cystic fibrosis.

Pseudomonas aeruginosa notoriously adapts to the airways of people with cystic fibrosis (CF), yet how infection-site biogeography and associated evolutionary processes vary as lifelong infections progress remains unclear. Here we test the hypothesis that early adaptations promoting aggregation influence evolutionary-genetic trajectories by examining longitudinal P. aeruginosa from the sinuses of six adults with CF. Highly host-adapted lineages harbored mutator genotypes displaying signatures of early genome degradation associated with recent host restriction. Using an advanced imaging technique (MiPACT-HCR [microbial identification after passive clarity technique]), we find population structure tracks with genome degradation, with the most host-adapted, genome-degraded P. aeruginosa (the mutators) residing in small, sparse aggregates. We propose that following initial adaptive evolution in larger populations under strong selection for aggregation, P. aeruginosa persists in small, fragmented populations that experience stronger effects of genetic drift. These conditions enrich for mutators and promote degenerative genome evolution. Our findings underscore the importance of infection-site biogeography to pathogen evolution.

Extracellular vesicles promote transkingdom nutrient transfer during viral-bacterial co-infection.

Extracellular vesicles (EVs) are increasingly appreciated as a mechanism of communication among cells that contribute to many physiological processes. Although EVs can promote either antiviral or proviral effects during viral infections, the role of EVs in virus-associated polymicrobial infections remains poorly defined. We report that EVs secreted from airway epithelial cells during respiratory viral infection promote secondary bacterial growth, including biofilm biogenesis, by Pseudomonas aeruginosa. Respiratory syncytial virus (RSV) increases the release of the host iron-binding protein transferrin on the extravesicular face of EVs, which interact with P. aeruginosa biofilms to transfer the nutrient iron and promote bacterial biofilm growth. Vesicular delivery of iron by transferrin more efficiently promotes P. aeruginosa biofilm growth than soluble holo-transferrin delivered alone. Our findings indicate that EVs are a nutrient source for secondary bacterial infections in the airways during viral infection and offer evidence of transkingdom communication in the setting of polymicrobial infections.

High terminal creatinine donors should not preclude simultaneous kidney and pancreas transplantation.

BACKGROUND: Simultaneous pancreas and kidney transplantation (SPK) in the setting of end-stage renal disease offers unmatched outcomes in insulin dependent diabetic patients. Donor pool expansion through the transplantation of kidneys with acute kidney injury (AKI) is controversial. METHODS: 59 SPK transplants were classified by presence of donor AKI, defined as donor terminal creatinine ≥ 1.5x the initial creatinine or donor terminal creatinine > 4.0 mg/dL. Endpoints included graft and patient survival, delayed graft function (DGF), serum creatinine, glomerular filtration rate (GFR), Hemoglobin A1c (HbA1c) and acute rejection. RESULTS: The donor AKI group (n = 35) had significantly higher rates of DGF (38 v. 9%, p = 0.01). There was no difference in creatinine or GFR at 1, 3, 6 and 12 months. HbA1c was comparable at 3, 6 and 12 months. There was no significant difference in the percentage of patients that required anti-diabetic agents after transplant (14 v. 4%, p = 0.56). CONCLUSIONS: We observed increased rates of DGF in SPK recipients with donor AKI. However, equivalent outcomes of pancreas and kidney function in both groups were observed.

Commercial insurance delays direct-acting antiviral treatment for hepatitis C kidney transplantation into uninfected recipients.

INTRODUCTION: The advent of direct-acting antivirals (DAAs) has created an avenue for transplantation of hepatitis C virus (HCV)-infected donors into uninfected recipients (D+/R-). The donor transmission of HCV is then countered by DAA administration during the post-operative period. However, initiation of DAA treatment is ultimately dictated by insurance companies. METHODS: A retrospective chart review of 52 D+/R- kidney recipients who underwent DAA treatment post-transplant was performed. Patients were grouped according to their prescription coverage plans, managed by either commercial or government pharmacy benefit managers (PBMs). RESULTS: Thirty-nine patients had government PBMs and 13 had commercial PBMs. Demographics were similar between the two groups. All patients developed HCV viremia, but cleared the virus after treatment with DAA. Patients with government PBMs were treated earlier compared to those with commercial PBMs (11 days vs 26 days, P = .01). Longer time to DAA initiation resulted in higher peak viral loads (ß = 0.39, R2  = .15, P = .01) and longer time to HCV viral load clearance (ß = 0.41, R2  = .17, P = .01). CONCLUSIONS: D+/R- transplantation offers patients an alternative strategy to increase access. However, treatment can be profoundly delayed by a third-party payer authorization process that may be subjecting patients to unnecessary risks and worsened outcomes.

Quantitative Framework for Model Evaluation in Microbiology Research Using Pseudomonas aeruginosa and Cystic Fibrosis Infection as a Test Case.

Laboratory models are a cornerstone of modern microbiology, but the accuracy of these models has not been systematically evaluated. As a result, researchers often choose models based on intuition or incomplete data. We propose a general quantitative framework to assess model accuracy from RNA sequencing data and use this framework to evaluate models of Pseudomonas aeruginosa cystic fibrosis (CF) lung infection. We found that an in vitro synthetic CF sputum medium model and a CF airway epithelial cell model had the highest genome-wide accuracy but underperformed on distinct functional categories, including porins and polyamine biosynthesis for the synthetic sputum medium and protein synthesis for the epithelial cell model. We identified 211 "elusive" genes that were not mimicked in a reference strain grown in any laboratory model but found that many were captured by using a clinical isolate. These methods provide researchers with an evidence-based foundation to select and improve laboratory models.IMPORTANCE Laboratory models have become a cornerstone of modern microbiology. However, the accuracy of even the most commonly used models has never been evaluated. Here, we propose a quantitative framework based on gene expression data to evaluate model performance and apply it to models of Pseudomonas aeruginosa cystic fibrosis lung infection. We discovered that these models captured different aspects of P. aeruginosa infection physiology, and we identify which functional categories are and are not captured by each model. These methods will provide researchers with a solid basis to choose among laboratory models depending on the scientific question of interest and will help improve existing experimental models.

Volatile fingerprinting of Pseudomonas aeruginosa and respiratory syncytial virus infection in an in vitro cystic fibrosis co-infection model.

Volatile molecules in exhaled breath represent potential biomarkers in the setting of infectious diseases, particularly those affecting the respiratory tract. In particular, Pseudomonas aeruginosa is a critically important respiratory pathogen in specific subsets of the population, such as those with cystic fibrosis (CF). Infections caused by P. aeruginosa can be particularly problematic when co-infection with respiratory syncytial virus (RSV) occurs, as this is correlated with the establishment of chronic P. aeruginosa infection. In the present study, we evaluate the volatile metabolites produced by P. aeruginosa (PAO1)-infected, RSV-infected, co-infected, or uninfected CF bronchial epithelial (CFBE) cells, in vitro. We identified a volatile metabolic signature that could discriminate between P. aeruginosa-infected and non-P. aeruginosa-infected CFBE with an area under the receiver operating characteristic curve (AUROC) of 0.850, using the machine learning algorithm random forest (RF). Although we could not discriminate between RSV-infected and non-RSV-infected CFBE (AUROC = 0.431), we note that sample classification probabilities for RSV-infected cell, generated using RF, were between those of uninfected CFBE and P. aeruginosa-infected CFBE, suggesting that RSV infection may result in a volatile metabolic profile that shares attributes with both of these groups. To more precisely elucidate the biological origins of the volatile metabolites that were discriminatory between P. aeruginosa-infected and non-P. aeruginosa-infected CFBE, we measured the volatile metabolites produced by P. aeruginosa grown in the absence of CFBE. Our findings suggest that the discriminatory metabolites produced likely result from the interaction of P. aeruginosa with the CFBE cells, rather than the metabolism of media components by the bacterium. Taken together, our findings support the notion that P. aeruginosa interacting with CFBE yields a particular volatile metabolic signature. Such a signature may have clinical utility in the monitoring of individuals with CF.

Pseudomonas aeruginosa Contact-Dependent Growth Inhibition Plays Dual Role in Host-Pathogen Interactions.

Microorganisms exist in a diverse ecosystem and have evolved many different mechanisms for sensing and influencing the polymicrobial environment around them, utilizing both diffusible and contact-dependent signals. Contact-dependent growth inhibition (CDI) is one such communication system employed by Gram-negative bacteria. In addition to CDI mediation of growth inhibition, recent studies have demonstrated CDI-mediated control of communal behaviors such as biofilm formation. We postulated that CDI may therefore play an active role in host-pathogen interactions, allowing invading strains to establish themselves at polymicrobial mucosal interfaces through competitive interactions while simultaneously facilitating pathogenic capabilities via CDI-mediated signaling. Here, we show that Pseudomonas aeruginosa produces two CDI systems capable of mediating competition under conditions of growth on a surface or in liquid. Furthermore, we demonstrated a novel role for these systems in contributing to virulence in acute infection models, likely via posttranscriptional regulation of beneficial behaviors. While we did not observe any role for the P. aeruginosa CDI systems in biofilm biogenesis, we did identify for the first time robust CDI-mediated competition during interaction with a mammalian host using a model of chronic respiratory tract infection, as well as evidence that CDI expression is maintained in chronic lung infections. These findings reveal a previously unappreciated role for CDI in host-pathogen interactions and emphasize their importance during infection. IMPORTANCE How bacteria compete and communicate with each other is an increasingly recognized aspect of microbial pathogenesis with a major impact on disease outcomes. Gram-negative bacteria have recently been shown to employ a contact-dependent toxin-antitoxin system to achieve both competition and regulation of their physiology. Here, we show that this system is vital for virulence in acute infection as well as for establishment of chronic infection in the multidrug-resistant pathogen Pseudomonas aeruginosa. Greater understanding of the mechanisms underlying bacterial virulence and infection is important for the development of effective therapeutics in the era of increasing antimicrobial resistance.

Bordetella adenylate cyclase toxin interacts with filamentous haemagglutinin to inhibit biofilm formation in vitro.

Bordetella pertussis, the causative agent of whooping cough, secretes and releases adenylate cyclase toxin (ACT), which is a protein bacterial toxin that targets host cells and disarms immune defenses. ACT binds filamentous haemagglutinin (FHA), a surface-displayed adhesin, and until now, the consequences of this interaction were unknown. A B. bronchiseptica mutant lacking ACT produced more biofilm than the parental strain; leading Irie et al. to propose the ACT-FHA interaction could be responsible for biofilm inhibition. Here we characterize the physical interaction of ACT with FHA and provide evidence linking that interaction to inhibition of biofilm in vitro. Exogenous ACT inhibits biofilm formation in a concentration-dependent manner and the N-terminal catalytic domain of ACT (AC domain) is necessary and sufficient for this inhibitory effect. AC Domain interacts with the C-terminal segment of FHA with ∼650 nM affinity. ACT does not inhibit biofilm formation by Bordetella lacking the mature C-terminal domain (MCD), suggesting the direct interaction between AC domain and the MCD is required for the inhibitory effect. Additionally, AC domain disrupts preformed biofilm on abiotic surfaces. The demonstrated inhibition of biofilm formation by a host-directed protein bacterial toxin represents a novel regulatory mechanism and identifies an unprecedented role for ACT.

Simultaneous Antibiofilm and Antiviral Activities of an Engineered Antimicrobial Peptide during Virus-Bacterium Coinfection.

Antimicrobial-resistant infections are an urgent public health threat, and development of novel antimicrobial therapies has been painstakingly slow. Polymicrobial infections are increasingly recognized as a significant source of severe disease and also contribute to reduced susceptibility to antimicrobials. Chronic infections also are characterized by their ability to resist clearance, which is commonly linked to the development of biofilms that are notorious for antimicrobial resistance. The use of engineered cationic antimicrobial peptides (eCAPs) is attractive due to the slow development of resistance to these fast-acting antimicrobials and their ability to kill multidrug-resistant clinical isolates, key elements for the success of novel antimicrobial agents. Here, we tested the ability of an eCAP, WLBU2, to disrupt recalcitrant Pseudomonas aeruginosa biofilms. WLBU2 was capable of significantly reducing biomass and viability of P. aeruginosa biofilms formed on airway epithelium and maintained activity during viral coinfection, a condition that confers extraordinary levels of antibiotic resistance. Biofilm disruption was achieved in short treatment times by permeabilization of bacterial membranes. Additionally, we observed simultaneous reduction of infectivity of the viral pathogen respiratory syncytial virus (RSV). WLBU2 is notable for its ability to maintain activity across a broad range of physiological conditions and showed negligible toxicity toward the airway epithelium, expanding its potential applications as an antimicrobial therapeutic. IMPORTANCE Antimicrobial-resistant infections are an urgent public health threat, making development of novel antimicrobials able to effectively treat these infections extremely important. Chronic and polymicrobial infections further complicate antimicrobial therapy, often through the development of microbial biofilms. Here, we describe the ability of an engineered antimicrobial peptide to disrupt biofilms formed by the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen Pseudomonas aeruginosa during coinfection with respiratory syncytial virus. We also observed antiviral activity, indicating the ability of engineered antimicrobial peptides to act as cross-kingdom single-molecule combination therapies.

Biochemical and Cellular Characterization and Inhibitor Discovery of Pseudomonas aeruginosa 15-Lipoxygenase.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial and chronic infections in immunocompromised patients. P. aeruginosa secretes a lipoxygenase, LoxA, but the biological role of this enzyme is currently unknown. LoxA is poorly similar in sequence to both soybean LOX-1 (s15-LOX-1) and human 15-LOX-1 (37 and 39%, respectively) yet has kinetics comparably fast versus those of s15-LOX-1 (at pH 6.5, Kcat = 181 ± 6 s(-1) and Kcat/KM = 16 ± 2 µM(-1) s(-1)). LoxA is capable of efficiently catalyzing the peroxidation of a broad range of free fatty acid (FA) substrates (e.g., AA and LA) with high positional specificity, indicating a 15-LOX. Its mechanism includes hydrogen atom abstraction [a kinetic isotope effect (KIE) of >30], yet LoxA is a poor catalyst against phosphoester FAs, suggesting that LoxA is not involved in membrane decomposition. LoxA also does not react with 5- or 15-HETEs, indicating poor involvement in lipoxin production. A LOX high-throughput screen of the LOPAC library yielded a variety of low-micromolar inhibitors; however, none selectively targeted LoxA over the human LOX isozymes. With respect to cellular activity, the level of LoxA expression is increased when P. aeruginosa undergoes the transition to a biofilm mode of growth, but LoxA is not required for biofilm growth on abiotic surfaces. However, LoxA does appear to be required for biofilm growth in association with the host airway epithelium, suggesting a role for LoxA in mediating bacterium-host interactions during colonization.

Engineered cationic antimicrobial peptide (eCAP) prevents Pseudomonas aeruginosa biofilm growth on airway epithelial cells.

OBJECTIVES: Chronic infections with the opportunistic pathogen Pseudomonas aeruginosa are responsible for the majority of the morbidity and mortality in patients with cystic fibrosis (CF). While P. aeruginosa infections may initially be treated successfully with standard antibiotics, chronic infections typically arise as bacteria transition to a biofilm mode of growth and acquire remarkable antimicrobial resistance. To address the critical need for novel antimicrobial therapeutics that can effectively suppress chronic bacterial infections in challenging physiological environments, such as the CF lung, we have rationally designed a de novo engineered cationic antimicrobial peptide, the 24-residue WLBU2, with broad-spectrum antibacterial activity for pan-drug-resistant P. aeruginosa in liquid culture. In the current study, we tested the hypothesis that WLBU2 also prevents P. aeruginosa biofilm growth. METHODS: Using abiotic and biotic biofilm assays, co-culturing P. aeruginosa with polarized human airway epithelial cells, we examined the ability of WLBU2 to prevent biofilm biogenesis alone and in combination with currently used antibiotics. RESULTS: We observed a dose-dependent reduction in biofilm growth on an abiotic surface and in association with CF airway epithelial cells. WLBU2 prevented P. aeruginosa biofilm formation when co-cultured with mucus-producing primary human CF airway epithelial cells and using CF clinical isolates of P. aeruginosa, even at low pH and high salt conditions that mimic the CF airway. When used in combination, WLBU2 significantly increases killing by the commonly used antibiotics tobramycin, ciprofloxacin, ceftazidime and meropenem. CONCLUSIONS: While other studies have demonstrated the ability of natural and synthetic antimicrobial peptides to prevent abiotic bacterial biofilm formation, the current studies for the first time demonstrate the effective peptide treatment of a biotic bacterial biofilm in a setting similar to the CF airway, and without negative effects on human airway epithelial cells, thus highlighting the unique potential of this engineered cationic antimicrobial peptide for treatment of human respiratory infections.

New Insight into Filamentous Hemagglutinin Secretion Reveals a Role for Full-Length FhaB in Bordetella Virulence.

UNLABELLED: Bordetella filamentous hemagglutinin (FHA), a primary component of acellular pertussis vaccines, contributes to virulence, but how it functions mechanistically is unclear. FHA is first synthesized as an ~370-kDa preproprotein called FhaB. Removal of an N-terminal signal peptide and a large C-terminal prodomain (PD) during secretion results in "mature" ~250-kDa FHA, which has been assumed to be the biologically active form of the protein. Deletion of two C-terminal subdomains of FhaB did not affect production of functional FHA, and the mutant strains were indistinguishable from wild-type bacteria for their ability to adhere to the lower respiratory tract and to suppress inflammation in the lungs of mice. However, the mutant strains, which produced altered FhaB molecules, were eliminated from the lower respiratory tract much faster than wild-type B. bronchiseptica, suggesting a defect in resistance to early immune-mediated clearance. Our results revealed, unexpectedly, that full-length FhaB plays a critical role in B. bronchiseptica persistence in the lower respiratory tract. IMPORTANCE: The Bordetella filamentous hemagglutinin (FHA) is a primary component of the acellular pertussis vaccine and an important virulence factor. FHA is initially produced as a large protein that is processed during secretion to the bacterial surface. As with most processed proteins, the mature form of FHA has been assumed to be the functional form of the protein. However, our results indicate that the full-length form plays an essential role in virulence in vivo. Furthermore, we have found that FHA contains intramolecular regulators of processing and that this control of processing is integral to its virulence activities. This report highlights the advantage of studying protein maturation and function simultaneously, as a role for the full-length form of FHA was evident only from in vivo infection studies and not from in vitro studies on the production or maturation of FHA or even from in vitro virulence-associated activity assays.

Cooperative roles for fimbria and filamentous hemagglutinin in Bordetella adherence and immune modulation.

UNLABELLED: Bordetella fimbriae (FIM) are generally considered to function as adhesins despite a lack of experimental evidence supporting this conclusion for Bordetella pertussis and evidence against a requirement for FIM in adherence of Bordetella bronchiseptica to mammalian cell lines. Using B. bronchiseptica and mice, we developed an in vivo adherence assay that revealed that FIM do function as critically important adhesins in the lower respiratory tract. In the first few days postinoculation, FIM-deficient B. bronchiseptica induced a more robust inflammatory response than wild-type bacteria did, suggesting that FIM, like filamentous hemagglutinin (FHA), allow B. bronchiseptica to suppress the innate immune response to infection. Localization analyses indicated that FIM are required for efficient attachment to airway epithelium, as bacteria lacking FIM localized to alveoli. FHA-deficient bacteria, in contrast, localized to airways. Bacteria unable to produce both FIM and FHA localized to alveoli and caused increased inflammation and histopathology identical to that caused by FIM-deficient bacteria, demonstrating that lack of FIM is epistatic to lack of FHA. Coinoculation experiments provided evidence that wild-type B. bronchiseptica suppresses inflammation locally within the respiratory tract and that both FHA and FIM are required for defense against clearance by the innate immune system. Altogether, our data suggest that FIM-mediated adherence to airway epithelium is a critical first step in Bordetella infection that allows FHA-dependent interactions to mediate tight adherence, suppression of inflammation, and resistance to inflammatory cell-mediated clearance. Our results suggest that mucosal antibodies capable of blocking FIM-mediated interactions could prevent bacterial colonization of the lower respiratory tract. IMPORTANCE: Although fimbriae (FIM) have been shown to be important mediators of adherence for many bacterial pathogens, there is surprisingly little experimental evidence supporting this role for Bordetella fimbria. Our results provide the first demonstration that Bordetella FIM function as adhesins in vivo, specifically to airway epithelium. Furthermore, our results suggest that FIM mediate initial interactions with airway epithelial cells that are followed by tight filamentous hemagglutinin (FHA)-mediated binding and that together, FIM and FHA allow Bordetella to suppress inflammation, leading to prolonged colonization. Given the shortcoming of the current acellular component pertussis (aP) vaccine in preventing colonization, these findings suggest that generation of antibodies capable of blocking FIM-mediated adherence could potentially prevent Bordetella colonization.