Infants with cystic fibrosis have altered fecal functional capacities with potential clinical and metabolic consequences.

BACKGROUND: Infants with cystic fibrosis (CF) suffer from gastrointestinal (GI) complications, including pancreatic insufficiency and intestinal inflammation, which have been associated with impaired nutrition and growth. Recent evidence identified altered fecal microbiota taxonomic compositions in infants with CF relative to healthy infants that were characterized by differences in the abundances of taxa associated with GI health and nutrition. Furthermore, these taxonomic differences were more pronounced in low length infants with CF, suggesting a potential link to linear growth failure. We hypothesized that these differences would entail shifts in the microbiome's functional capacities that could contribute to inflammation and nutritional failure in infants with CF. RESULTS: To test this hypothesis, we compared fecal microbial metagenomic content between healthy infants and infants with CF, supplemented with an analysis of fecal metabolomes in infants with CF. We identified notable differences in CF fecal microbial functional capacities, including metabolic and environmental response functions, compared to healthy infants that intensified during the first year of life. A machine learning-based longitudinal metagenomic age analysis of healthy and CF fecal metagenomic functional profiles further demonstrated that these differences are characterized by a CF-associated delay in the development of these functional capacities. Moreover, we found metagenomic differences in functions related to metabolism among infants with CF that were associated with diet and antibiotic exposure, and identified several taxa as potential drivers of these functional differences. An integrated metagenomic and metabolomic analysis further revealed that abundances of several fecal GI metabolites important for nutrient absorption, including three bile acids, correlated with specific microbes in infants with CF. CONCLUSIONS: Our results highlight several metagenomic and metabolomic factors, including bile acids and other microbial metabolites, that may impact nutrition, growth, and GI health in infants with CF. These factors could serve as promising avenues for novel microbiome-based therapeutics to improve health outcomes in these infants.

Maintenance tobramycin primarily affects untargeted bacteria in the CF sputum microbiome.

RATIONALE: The most common antibiotic used to treat people with cystic fibrosis (PWCF) is inhaled tobramycin, administered as maintenance therapy for chronic Pseudomonas aeruginosa lung infections. While the effects of inhaled tobramycin on P. aeruginosa abundance and lung function diminish with continued therapy, this maintenance treatment is known to improve long-term outcomes, underscoring how little is known about why antibiotics work in CF infections, what their effects are on complex CF sputum microbiomes and how to improve these treatments. OBJECTIVES: To rigorously define the effect of maintenance tobramycin on CF sputum microbiome characteristics. METHODS AND MEASUREMENTS: We collected sputum from 30 PWCF at standardised times before, during and after a single month-long course of maintenance inhaled tobramycin. We used traditional culture, quantitative PCR and metagenomic sequencing to define the dynamic effects of this treatment on sputum microbiomes, including abundance changes in both clinically targeted and untargeted bacteria, as well as functional gene categories. MAIN RESULTS: CF sputum microbiota changed most markedly by 1 week of antibiotic therapy and plateaued thereafter, and this shift was largely driven by changes in non-dominant taxa. The genetically conferred functional capacities (ie, metagenomes) of subjects' sputum communities changed little with antibiotic perturbation, despite taxonomic shifts, suggesting functional redundancy within the CF sputum microbiome. CONCLUSIONS: Maintenance treatment with inhaled tobramycin, an antibiotic with demonstrated long-term mortality benefit, primarily impacted clinically untargeted bacteria in CF sputum, highlighting the importance of monitoring the non-canonical effects of antibiotics and other treatments to accurately define and improve their clinical impact.

CFTR dysregulation drives active selection of the gut microbiome.

Patients with cystic fibrosis (CF) have altered fecal microbiomes compared to those of healthy controls. The magnitude of this dysbiosis correlates with measures of CF gastrointestinal (GI) disease, including GI inflammation and nutrient malabsorption. However, whether this dysbiosis is caused by mutations in the CFTR gene, the underlying defect in CF, or whether CF-associated dysbiosis augments GI disease was not clear. To test the relationships between CFTR dysfunction, microbes, and intestinal health, we established a germ-free (GF) CF mouse model and demonstrated that CFTR gene mutations are sufficient to alter the GI microbiome. Furthermore, flow cytometric analysis demonstrated that colonized CF mice have increased mesenteric lymph node and spleen TH17+ cells compared with non-CF mice, suggesting that CFTR defects alter adaptive immune responses. Our findings demonstrate that CFTR mutations modulate both the host adaptive immune response and the intestinal microbiome.

Fecal dysbiosis in infants with cystic fibrosis is associated with early linear growth failure.

Most infants with cystic fibrosis (CF) have pancreatic exocrine insufficiency that results in nutrient malabsorption and requires oral pancreatic enzyme replacement. Newborn screening for CF has enabled earlier diagnosis, nutritional intervention and enzyme replacement for these infants, allowing most infants with CF to achieve their weight goals by 12 months of age1. Nevertheless, most infants with CF continue to have poor linear growth during their first year of life1. Although this early linear growth failure is associated with worse long-term respiratory function and survival2,3, the determinants of body length in infants with CF have not been defined. Several characteristics of the CF gastrointestinal (GI) tract, including inflammation, maldigestion and malabsorption, may promote intestinal dysbiosis4,5. As GI microbiome activities are known to affect endocrine functions6,7, the intestinal microbiome of infants with CF may also impact growth. We identified an early, progressive fecal dysbiosis that distinguished infants with CF and low length from infants with CF and normal length. This dysbiosis included altered abundances of taxa that perform functions that are important for GI health, nutrient harvest and growth hormone signaling, including decreased abundance of Bacteroidetes and increased abundance of Proteobacteria. Thus, the GI microbiota represent a potential therapeutic target for the correction of low linear growth in infants with CF.

Human and Extracellular DNA Depletion for Metagenomic Analysis of Complex Clinical Infection Samples Yields Optimized Viable Microbiome Profiles.

Metagenomic sequencing is a promising approach for identifying and characterizing organisms and their functional characteristics in complex, polymicrobial infections, such as airway infections in people with cystic fibrosis. These analyses are often hampered, however, by overwhelming quantities of human DNA, yielding only a small proportion of microbial reads for analysis. In addition, many abundant microbes in respiratory samples can produce large quantities of extracellular bacterial DNA originating either from biofilms or dead cells. We describe a method for simultaneously depleting DNA from intact human cells and extracellular DNA (human and bacterial) in sputum, using selective lysis of eukaryotic cells and endonuclease digestion. We show that this method increases microbial sequencing depth and, consequently, both the number of taxa detected and coverage of individual genes such as those involved in antibiotic resistance. This finding underscores the substantial impact of DNA from sources other than live bacteria in microbiological analyses of complex, chronic infection specimens.

Adaptation of commensal proliferating Escherichia coli to the intestinal tract of young children with cystic fibrosis.

The mature human gut microbiota is established during the first years of life, and altered intestinal microbiomes have been associated with several human health disorders. Escherichia coli usually represents less than 1% of the human intestinal microbiome, whereas in cystic fibrosis (CF), greater than 50% relative abundance is common and correlates with intestinal inflammation and fecal fat malabsorption. Despite the proliferation of E. coli and other Proteobacteria in conditions involving chronic gastrointestinal tract inflammation, little is known about adaptation of specific characteristics associated with microbiota clonal expansion. We show that E. coli isolated from fecal samples of young children with CF has adapted to growth on glycerol, a major component of fecal fat. E. coli isolates from different CF patients demonstrate an increased growth rate in the presence of glycerol compared with E. coli from healthy controls, and unrelated CF E. coli strains have independently acquired this growth trait. Furthermore, CF and control E. coli isolates have differential gene expression when grown in minimal media with glycerol as the sole carbon source. While CF isolates display a growth-promoting transcriptional profile, control isolates engage stress and stationary-phase programs, which likely results in slower growth rates. Our results indicate that there is selection of unique characteristics within the microbiome of individuals with CF, which could contribute to individual disease outcomes.

Clinical and Fecal Microbial Changes With Diet Therapy in Active Inflammatory Bowel Disease.

GOAL: To determine the effect of the specific carbohydrate diet (SCD) on active inflammatory bowel disease (IBD). BACKGROUND: IBD is a chronic idiopathic inflammatory intestinal disorder associated with fecal dysbiosis. Diet is a potential therapeutic option for IBD based on the hypothesis that changing the fecal dysbiosis could decrease intestinal inflammation. STUDY: Pediatric patients with mild to moderate IBD defined by pediatric Crohn's disease activity index (PCDAI 10-45) or pediatric ulcerative colitis activity index (PUCAI 10-65) were enrolled into a prospective study of the SCD. Patients started SCD with follow-up evaluations at 2, 4, 8, and 12 weeks. PCDAI/PUCAI, laboratory studies were assessed. RESULTS: Twelve patients, ages 10 to 17 years, were enrolled. Mean PCDAI decreased from 28.1±8.8 to 4.6±10.3 at 12 weeks. Mean PUCAI decreased from 28.3±23.1 to 6.7±11.6 at 12 weeks. Dietary therapy was ineffective for 2 patients while 2 individuals were unable to maintain the diet. Mean C-reactive protein decreased from 24.1±22.3 to 7.1±0.4 mg/L at 12 weeks in Seattle Cohort (nL<8.0 mg/L) and decreased from 20.7±10.9 to 4.8±4.5 mg/L at 12 weeks in Atlanta Cohort (nL<4.9 mg/L). Stool microbiome analysis showed a distinctive dysbiosis for each individual in most prediet microbiomes with significant changes in microbial composition after dietary change. CONCLUSIONS: SCD therapy in IBD is associated with clinical and laboratory improvements as well as concomitant changes in the fecal microbiome. Further prospective studies are required to fully assess the safety and efficacy of dietary therapy in patients with IBD.

GUTSS: An Alignment-Free Sequence Comparison Method for Use in Human Intestinal Microbiome and Fecal Microbiota Transplantation Analysis.

BACKGROUND: Comparative analysis of gut microbiomes in clinical studies of human diseases typically rely on identification and quantification of species or genes. In addition to exploring specific functional characteristics of the microbiome and potential significance of species diversity or expansion, microbiome similarity is also calculated to study change in response to therapies directed at altering the microbiome. Established ecological measures of similarity can be constructed from species abundances, however methods for calculating these commonly used ecological measures of similarity directly from whole genome shotgun (WGS) metagenomic sequence are lacking. RESULTS: We present an alignment-free method for calculating similarity of WGS metagenomic sequences that is analogous to the Bray-Curtis index for species, implemented by the General Utility for Testing Sequence Similarity (GUTSS) software application. This method was applied to intestinal microbiomes of healthy young children to measure developmental changes toward an adult microbiome during the first 3 years of life. We also calculate similarity of donor and recipient microbiomes to measure establishment, or engraftment, of donor microbiota in fecal microbiota transplantation (FMT) studies focused on mild to moderate Crohn's disease. We show how a relative index of similarity to donor can be calculated as a measure of change in a patient's microbiome toward that of the donor in response to FMT. CONCLUSION: Because clinical efficacy of the transplant procedure cannot be fully evaluated without analysis methods to quantify actual FMT engraftment, we developed a method for detecting change in the gut microbiome that is independent of species identification and database bias, sensitive to changes in relative abundance of the microbial constituents, and can be formulated as an index for correlating engraftment success with clinical measures of disease. More generally, this method may be applied to clinical evaluation of human microbiomes and provide potential diagnostic determination of individuals who may be candidates for specific therapies directed at alteration of the microbiome.

Metagenomic evidence for taxonomic dysbiosis and functional imbalance in the gastrointestinal tracts of children with cystic fibrosis.

Cystic fibrosis (CF) results in inflammation, malabsorption of fats and other nutrients, and obstruction in the gastrointestinal (GI) tract, yet the mechanisms linking these disease manifestations to microbiome composition remain largely unexplored. Here we used metagenomic analysis to systematically characterize fecal microbiomes of children with and without CF, demonstrating marked CF-associated taxonomic dysbiosis and functional imbalance. We further showed that these taxonomic and functional shifts were especially pronounced in young children with CF and diminished with age. Importantly, the resulting dysbiotic microbiomes had significantly altered capacities for lipid metabolism, including decreased capacity for overall fatty acid biosynthesis and increased capacity for degrading anti-inflammatory short-chain fatty acids. Notably, these functional differences correlated with fecal measures of fat malabsorption and inflammation. Combined, these results suggest that enteric fat abundance selects for pro-inflammatory GI microbiota in young children with CF, offering novel strategies for improving the health of children with CF-associated fat malabsorption.

Low Level Engraftment and Improvement following a Single Colonoscopic Administration of Fecal Microbiota to Patients with Ulcerative Colitis.

OBJECTIVE: Fecal microbiota transplantation (FMT) is an investigational treatment for diseases thought to involve alterations in the intestinal microbiota including ulcerative colitis (UC). Case reports have described therapeutic benefit of FMT in patients with UC, possibly due to changes in the microbiota. We measured the degree to which the transplanted microbiota engraft following FMT in patients with UC using a donor similarity index (DSI). METHODS: Seven patients with mild to moderate UC (UC disease activity index scores 3-10) received a single colonoscopic administration of FMT. Metagenomic sequence data from stool were analyzed using an alignment-free comparison tool, to measure the DSI, and a phylogenetic analysis tool, to characterize taxonomic changes. Clinical, endoscopic, histologic, and fecal calprotectin outcome measures were also collected. RESULTS: One of 5 patients from whom sequencing data were available achieved the primary endpoint of 50% donor similarity at week 4; an additional 2 patients achieved 40% donor similarity. One patient with 40% donor similarity achieved clinical and histologic remission 1 month after FMT. However, these were lost by 2-3 months, and loss correlated with a decrease in DSI. The remaining patients did not demonstrate clinical response or remission. Histology scores improved in all but 1 patient. No patients remained in remission at 3 months after FMT. CONCLUSIONS: Following a single colonoscopic fecal transplant, a DSI of 40-50% is achieved in about two-thirds of recipients. This level of engraftment correlated with a temporary clinical improvement in only 1/5 patients. Larger sample sizes could further validate this method for measuring engraftment, and changes in transplant frequency or method might improve microbiota engraftment and efficacy. TRIAL REGISTRATION: ClinicalTrials.gov NCT01742754.

Regional Isolation Drives Bacterial Diversification within Cystic Fibrosis Lungs.

Bacterial lineages that chronically infect cystic fibrosis (CF) patients genetically diversify during infection. However, the mechanisms driving diversification are unknown. By dissecting ten CF lung pairs and studying ∼12,000 regional isolates, we were able to investigate whether clonally related Pseudomonas aeruginosa inhabiting different lung regions evolve independently and differ functionally. Phylogenetic analysis of genome sequences showed that regional isolation of P. aeruginosa drives divergent evolution. We investigated the consequences of regional evolution by studying isolates from mildly and severely diseased lung regions and found evolved differences in bacterial nutritional requirements, host defense and antibiotic resistance, and virulence due to hyperactivity of the type 3 secretion system. These findings suggest that bacterial intermixing is limited in CF lungs and that regional selective pressures may markedly differ. The findings also may explain how specialized bacterial variants arise during infection and raise the possibility that pathogen diversification occurs in other chronic infections characterized by spatially heterogeneous conditions.

Fecal microbial transplant effect on clinical outcomes and fecal microbiome in active Crohn's disease.

BACKGROUND: Crohn's disease (CD) is a chronic idiopathic inflammatory intestinal disorder associated with fecal dysbiosis. Fecal microbial transplant (FMT) is a potential therapeutic option for individuals with CD based on the hypothesis that changing the fecal dysbiosis could promote less intestinal inflammation. METHODS: Nine patients, aged 12 to 19 years, with mild-to-moderate symptoms defined by Pediatric Crohn's Disease Activity Index (PCDAI of 10-29) were enrolled into a prospective open-label study of FMT in CD (FDA IND 14942). Patients received FMT by nasogastric tube with follow-up evaluations at 2, 6, and 12 weeks. PCDAI, C-reactive protein, and fecal calprotectin were evaluated at each study visit. RESULTS: All reported adverse events were graded as mild except for 1 individual who reported moderate abdominal pain after FMT. All adverse events were self-limiting. Metagenomic evaluation of stool microbiome indicated evidence of FMT engraftment in 7 of 9 patients. The mean PCDAI score improved with patients having a baseline of 19.7 ± 7.2, with improvement at 2 weeks to 6.4 ± 6.6 and at 6 weeks to 8.6 ± 4.9. Based on PCDAI, 7 of 9 patients were in remission at 2 weeks and 5 of 9 patients who did not receive additional medical therapy were in remission at 6 and 12 weeks. No or modest improvement was seen in patients who did not engraft or whose microbiome was most similar to their donor. CONCLUSIONS: This is the first study to demonstrate that FMT for CD may be a possible therapeutic option for CD. Further prospective studies are required to fully assess the safety and efficacy of the FMT in patients with CD.

Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets.

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 µg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.

Escherichia coli dysbiosis correlates with gastrointestinal dysfunction in children with cystic fibrosis.

Cystic fibrosis gastrointestinal disease includes nutrient malabsorption and intestinal inflammation. We show that the abundances of Escherichia coli in fecal microbiota were significantly higher in young children with cystic fibrosis than in controls and correlated with fecal measures of nutrient malabsorption and inflammation, suggesting that E. coli could contribute to cystic fibrosis gastrointestinal dysfunction.

Strain-dependent diversity in the Pseudomonas aeruginosa quorum-sensing regulon.

Quorum sensing allows bacteria to sense and respond to changes in population density. Acyl-homoserine lactones serve as quorum-sensing signals for many Proteobacteria, and acyl-homoserine lactone signaling is known to control cooperative activities. Quorum-controlled activities vary from one species to another. Quorum-sensing controls a constellation of genes in the opportunistic pathogen Pseudomonas aeruginosa, which thrives in a number of habitats ranging from soil and water to animal hosts. We hypothesized that there would be significant variation in quorum-sensing regulons among strains of P. aeruginosa isolated from different habitats and that differences in the quorum-sensing regulons might reveal insights about the ecology of P. aeruginosa. As a test of our hypothesis we used RNA-seq to identify quorum-controlled genes in seven P. aeruginosa isolates of diverse origins. Although our approach certainly overlooks some quorum-sensing-regulated genes we found a shared set of genes, i.e., a core quorum-controlled gene set, and we identified distinct, strain-variable sets of quorum-controlled genes, i.e., accessory genes. Some quorum-controlled genes in some strains were not present in the genomes of other strains. We detected a correlation between traits encoded by some genes in the strain-variable subsets of the quorum regulons and the ecology of the isolates. These findings indicate a role for quorum sensing in extension of the range of habitats in which a species can thrive. This study also provides a framework for understanding the molecular mechanisms by which quorum-sensing systems operate, the evolutionary pressures by which they are maintained, and their importance in disparate ecological contexts.

Functional genetic screen of human diversity reveals that a methionine salvage enzyme regulates inflammatory cell death.

Genome-wide association studies can identify common differences that contribute to human phenotypic diversity and disease. When genome-wide association studies are combined with approaches that test how variants alter physiology, biological insights can emerge. Here, we used such an approach to reveal regulation of cell death by the methionine salvage pathway. A common SNP associated with reduced expression of a putative methionine salvage pathway dehydratase, apoptotic protease activating factor 1 (APAF1)-interacting protein (APIP), was associated with increased caspase-1-mediated cell death in response to Salmonella. The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5'-methylthioadenosine. Reducing expression of APIP or exogenous addition of 5'-methylthioadenosine increased Salmonellae-induced cell death. Consistent with APIP originally being identified as an inhibitor of caspase-9-dependent apoptosis, the same allele was also associated with increased sensitivity to the chemotherapeutic agent carboplatin. Our results show that common human variation affecting expression of a single gene can alter susceptibility to two distinct cell death programs. Furthermore, the same allele that promotes cell death is associated with improved survival of individuals with systemic inflammatory response syndrome, suggesting a possible evolutionary pressure that may explain the geographic pattern observed for the frequency of this SNP. Our study shows that in vitro association screens of disease-related traits can not only reveal human genetic differences that contribute to disease but also provide unexpected insights into cell biology.

A genome-wide in vitro bacterial-infection screen reveals human variation in the host response associated with inflammatory disease.

Recent progress in cataloguing common genetic variation has made possible genome-wide studies that are beginning to elucidate the causes and consequences of our genetic differences. Approaches that provide a mechanistic understanding of how genetic variants function to alter disease susceptibility and why they were substrates of natural selection would complement other approaches to human-genome analysis. Here we use a novel cell-based screen of bacterial infection to identify human variation in Salmonella-induced cell death. A loss-of-function allele of CARD8, a reported inhibitor of the proinflammatory protease caspase-1, was associated with increased cell death in vitro (p = 0.013). The validity of this association was demonstrated through overexpression of alternative alleles and RNA interference in cells of varying genotype. Comparison of mammalian CARD8 orthologs and examination of variation among different human populations suggest that the increase in infectious-disease burden associated with larger animal groups (i.e., herds and colonies), and possibly human population expansion, may have naturally selected for loss of CARD8. We also find that the loss-of-function CARD8 allele shows a modest association with an increased risk of systemic inflammatory response syndrome in a small study (p = 0.05). Therefore, a by-product of the selected benefit of loss of CARD8 could be increased inflammatory diseases. These results demonstrate the utility of genome-wide cell-based association screens with microbes in the identification of naturally selected variants that can impact human health.

Determination and comparison of the Francisella tularensis subsp.novicida U112 proteome to other bacterial proteomes.

The proteins expressed by Francisella tularensis subsp. novicida U112 grown to midexponential phase were surveyed by nanoLC-tandem mass spectrometry (LC-MS/MS). To improve annotation of the genome and develop a technology to provide high-throughput analysis of the Francisella proteome in multiple conditions, we sought to establish a fast and simple analysis that would reduce as much as possible the false discovery rate. Our survey detected expression of 63.0% of the predicted proteome from the stable condition of growth in rich medium available at (www.francisella.org). On the basis of detection of essential proteins, we estimated coverage to be approximately 80% of the actual expressed proteome. This suggests that no less than 70% of the proteins could be expressed in this condition. This analysis revealed two previously unidentified protein coding open reading frames and validated 50% of the proteins annotated as hypothetical. On the basis of results of the screen to detect essential proteins, not all proteins expressed provide a measurable contribution to F.t. novicida growth in this condition. Comparison of this protein profile with other profiles previously published suggested that the genome size and number of genes involved in regulation have little effect on the number of proteins expressed in a given stable condition.

A Francisella mutant in lipid A carbohydrate modification elicits protective immunity.

Francisella tularensis (Ft) is a highly infectious gram-negative bacterium and the causative agent of the human disease tularemia. Ft is designated a class A select agent by the Centers for Disease Control and Prevention. Human clinical isolates of Ft produce lipid A of similar structure to Ft subspecies novicida (Fn), a pathogen of mice. We identified three enzymes required for Fn lipid A carbohydrate modifications, specifically the presence of mannose (flmF1), galactosamine (flmF2), or both carbohydrates (flmK). Mutants lacking either galactosamine (flmF2) or galactosamine/mannose (flmK) addition to their lipid A were attenuated in mice by both pulmonary and subcutaneous routes of infection. In addition, aerosolization of the mutants (flmF2 and flmK) provided protection against challenge with wild-type (WT) Fn, whereas subcutaneous administration of only the flmK mutant provided protection from challenge with WT Fn. Furthermore, infection of an alveolar macrophage cell line by the flmK mutant induced higher levels of tumor necrosis factor-alpha (TNF-alpha) and macrophage inhibitory protein-2 (MIP-2) when compared to infection with WT Fn. Bone marrow-derived macrophages (BMMø) from Toll-like receptor 4 (TLR4) and TLR2/4 knockout mice infected with the flmK mutant also produced significantly higher amounts of interleukin-6 (IL-6) and MIP-2 than BMMø infected with WT Fn. However, production of IL-6 and MIP-2 was undetectable in BMMø from MyD88(-/-) mice infected with either strain. MyD88(-/-) mice were also susceptible to flmK mutant infection. We hypothesize that the ability of the flmK mutant to activate pro-inflammatory cytokine/chemokine production and innate immune responses mediated by the MyD88 signaling pathway may be responsible for its attenuation, leading to the induction of protective immunity by this mutant.

Type IV pili-mediated secretion modulates Francisella virulence.

Francisella tularensis are the causative agent of the zoonotic disease, tularaemia. Among four F. tularensis subspecies, ssp. novicida (F. novicida) is pathogenic only for immunocompromised individuals, while all four subspecies are pathogenic for mice. This study utilized proteomic and bioinformatic approaches to identify seven F. novicida secreted proteins and the corresponding Type IV pilus (T4P) secretion system. The secreted proteins were predicted to encode two chitinases, a chitin binding protein, a protease (PepO), and a beta-glucosidase (BglX). The transcription of F. novicida pepO and bglX was regulated by the virulence regulator MglA. Intradermal infection of mice with F. novicida mutants defective in T4P secretion system or PepO resulted in enhanced F. novicida spread to systemic sites. Infection with F. novicida pepO mutants also resulted in increased neutrophil infiltration into the mouse airways. PepO is a zinc protease that is homologous to mammalian endothelin-converting enzyme ECE-1. Therefore, secretion of PepO likely results in increased production of endothelin and increased vasoconstriction at the infection site in skin that limits the F. novicida spread. Francisella human pathogenic strains contain a mutation in pepO predicted to abolish its secretion. Loss of PepO function may have contributed to evolution of highly virulent Francisellae.