Pseudomonas aeruginosa Lipid A Structural Variants Induce Altered Immune Responses.

Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection that is not seen in any other disease state. Lipid A, the membrane anchor of LPS (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent Toll-like receptor 4 (TLR4) agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of the P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human BAL fluid. This structure triggers increased proinflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CF transmembrane conductance regulator function. It is interesting that there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lacks PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.

Remodeling of Lipid A in Pseudomonas syringae pv. phaseolicola In Vitro.

Pseudomonas species infect a variety of organisms, including mammals and plants. Mammalian pathogens of the Pseudomonas family modify their lipid A during host entry to evade immune responses and to create an effective barrier against different environments, for example by removal of primary acyl chains, addition of phosphoethanolamine (P-EtN) to primary phosphates, and hydroxylation of secondary acyl chains. For Pseudomonas syringae pv. phaseolicola (Pph) 1448A, an economically important pathogen of beans, we observed similar lipid A modifications by mass spectrometric analysis. Therefore, we investigated predicted proteomes of various plant-associated Pseudomonas spp. for putative lipid A-modifying proteins using the well-studied mammalian pathogen Pseudomonas aeruginosa as a reference. We generated isogenic mutant strains of candidate genes and analyzed their lipid A. We show that the function of PagL, LpxO, and EptA is generally conserved in Pph 1448A. PagL-mediated de-acylation occurs at the distal glucosamine, whereas LpxO hydroxylates the secondary acyl chain on the distal glucosamine. The addition of P-EtN catalyzed by EptA occurs at both phosphates of lipid A. Our study characterizes lipid A modifications in vitro and provides a useful set of mutant strains relevant for further functional studies on lipid A modifications in Pph 1448A.

Whole-Genome Sequencing To Identify Drivers of Carbapenem-Resistant Klebsiella pneumoniae Transmission within and between Regional Long-Term Acute-Care Hospitals.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an antibiotic resistance threat of the highest priority. Given the limited treatment options for this multidrug-resistant organism (MDRO), there is an urgent need for targeted strategies to prevent transmission. Here, we applied whole-genome sequencing to a comprehensive collection of clinical isolates to reconstruct regional transmission pathways and analyzed this transmission network in the context of statewide patient transfer data and patient-level clinical data to identify drivers of regional transmission. We found that high regional CRKP burdens were due to a small number of regional introductions, with subsequent regional proliferation occurring via patient transfers among health care facilities. While CRKP was predicted to have been imported into each facility multiple times, there was substantial variation in the ratio of intrafacility transmission events per importation, indicating that amplification occurs unevenly across regional facilities. While myriad factors likely influence intrafacility transmission rates, an understudied one is the potential for clinical characteristics of colonized and infected patients to influence their propensity for transmission. Supporting the contribution of high-risk patients to elevated transmission rates, we observed that patients colonized and infected with CRKP in high-transmission facilities had higher rates of carbapenem use, malnutrition, and dialysis and were older. This report highlights the potential for regional infection prevention efforts that are grounded in genomic epidemiology to identify the patients and facilities that make the greatest contribution to regional MDRO prevalence, thereby facilitating the design of precision interventions of maximal impact.

Microbial Lineages in Sarcoidosis. A Metagenomic Analysis Tailored for Low-Microbial Content Samples.

RATIONALE: The etiology of sarcoidosis is unknown, but microbial agents are suspected as triggers. OBJECTIVES: We sought to identify bacterial, fungal, or viral lineages in specimens from patients with sarcoidosis enriched relative to control subjects using metagenomic DNA sequencing. Because DNA from environmental contamination contributes disproportionately to samples with low authentic microbial content, we developed improved methods for filtering environmental contamination. METHODS: We analyzed specimens from subjects with sarcoidosis (n = 93), control subjects without sarcoidosis (n = 72), and various environmental controls (n = 150). Sarcoidosis specimens consisted of two independent sets of formalin-fixed, paraffin-embedded lymph node biopsies, BAL, Kveim reagent, and fresh granulomatous spleen from a patient with sarcoidosis. All specimens were analyzed by bacterial 16S and fungal internal transcribed spacer ribosomal RNA gene sequencing. In addition, BAL was analyzed by shotgun sequencing of fractions enriched for viral particles, and Kveim and spleen were subjected to whole-genome shotgun sequencing. MEASUREMENTS AND MAIN RESULTS: In one tissue set, fungi in the Cladosporiaceae family were enriched in sarcoidosis compared with nonsarcoidosis tissues; in the other tissue set, we detected enrichment of several bacterial lineages in sarcoidosis but not Cladosporiaceae. BAL showed limited enrichment of Aspergillus fungi. Several microbial lineages were detected in Kveim and spleen, including Cladosporium. No microbial lineage was enriched in more than one sample type after correction for multiple comparisons. CONCLUSIONS: Metagenomic sequencing revealed enrichment of microbes in single types of sarcoidosis samples but limited concordance across sample types. Statistical analysis accounting for environmental contamination was essential to avoiding false positives.

Divergent Pseudomonas aeruginosa LpxO enzymes perform site-specific lipid A 2-hydroxylation.

Pseudomonas aeruginosa can survive in a myriad of environments, partially due to modifications of its lipid A, the membrane anchor of lipopolysaccharide. We previously demonstrated that divergent late acyltransferase paralogs, HtrB1 and HtrB2, add acyloxyacyl laurate to lipid A 2- and 2'-acyl chains, respectively. The genome of P. aeruginosa also has genes which encode two dioxygenase enzymes, LpxO1 and LpxO2, that individually hydroxylate a specific secondary laurate. LpxO1 acts on the 2'-acyloxyacyl laurate (added by HtrB2), whereas LpxO2 acts on the 2-acyloxyacyl laurate (added by HtrB1) in a site-specific manner. Furthermore, while both enzyme pairs are evolutionarily linked, phylogenomic analysis suggests the LpxO1/HtrB2 enzyme pair as being of ancestral origin, present throughout the Pseudomonas lineage, whereas the LpxO2/HtrB1 enzyme pair likely arose via horizontal gene transfer and has been retained in P. aeruginosa over time. Using a murine pulmonary infection model, we showed that both LpxO1 and LpxO2 enzymes are functional in vivo, as direct analysis of in vivo lipid A structure from bronchoalveolar lavage fluid revealed 2-hydroxylated lipid A. Gene expression analysis reveals increased lpxO2 but unchanged lpxO1 expression in vivo, suggesting differential regulation of these enzymes during infection. We also demonstrate that loss-of-function mutations arise in lpxO1 and lpxO2 during chronic lung infection in people with cystic fibrosis (CF), indicating a potential role for pathogenesis and airway adaptation. Collectively, our study characterizes lipid A 2-hydroxylation during P. aeruginosa airway infection that is regulated by two distinct lipid A dioxygenase enzymes.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes severe infection in hospitalized and chronically ill individuals. During infection, P. aeruginosa undergoes adaptive changes to evade host defenses and therapeutic interventions, increasing mortality and morbidity. Lipid A structural alteration is one such change that P. aeruginosa isolates undergo during chronic lung infection in CF. Investigating genetic drivers of this lipid A structural variation is crucial in understanding P. aeruginosa adaptation during infection. Here, we describe two lipid A dioxygenases with acyl-chain site specificity, each with different evolutionary origins. Further, we show that loss of function in these enzymes occurs in CF clinical isolates, suggesting a potential pathoadaptive phenotype. Studying these bacterial adaptations provides insight into selection pressures of the CF airway on P. aeruginosa phenotypes that persist during chronic infection. Understanding these adaptive changes may ultimately provide clinicians better control over bacterial populations during chronic infection.

Asymptomatic Esophageal Eosinophilia in an 11-Year-Old with Severe Persistent Asthma.

Asymptomatic esophageal eosinophilia (aEE) is a rare presentation, where patients have increased eosinophils in esophageal mucosa but lack any esophagus-related symptoms. Cases of aEE have only been documented in adults, and little is known about its clinical significance and whether treatment is warranted. We report a case of an 11-year-old patient with uncontrolled severe persistent asthma who underwent flexible bronchoscopy and upper endoscopy as a part of complete aerodigestive evaluation. Elevated intraepithelial eosinophils in the esophageal mucosa were noted, suggesting an aEE-like presentation. This case documents a pediatric patient with aEE and highlights the importance of combined aerodigestive assessment with pulmonology and gastroenterology teams for the evaluation of severe asthma.

Spatial lipidomics reveals biased phospholipid remodeling in acute Pseudomonas lung infection.

Pseudomonas aeruginosa (Pa) is a pathogen causing chronic pulmonary infections in patients with cystic fibrosis (CF). Manipulation of lipids is an important feature of Pa infection and on a tissue-level scale is poorly understood. Using a mouse model of acute Pa pulmonary infection, we explored the whole-lung phospholipid response using mass spectrometry imaging (MSI) and spatial lipidomics. Using a histology-driven analysis, we isolated airways and parenchyma from both mock- and Pa-infected lungs and used systems biology tools to identify enriched metabolic pathways from the differential phospholipid identities. Infection was associated with a set of 26 ions, with 11 unique to parenchyma and 6 unique to airways. Acyl remodeling was differentially enriched in infected parenchyma as the predominant biological function. These functions correlated with markers of polymorphonuclear (PMN) cell influx, a defining feature of the lung response to Pa infection, implicating enzymes active in phospholipid remodeling.

Genomic and Functional Characterization of Longitudinal Pseudomonas aeruginosa Isolates from Young Patients with Cystic Fibrosis.

Individuals with cystic fibrosis (CF) suffer from frequent and recurring microbial airway infections. The Gram-negative bacterium Pseudomonas aeruginosa is one of the most common organisms isolated from CF patient airways. P. aeruginosa establishes chronic infections that persist throughout a patient's lifetime and is a major cause of morbidity and mortality. Throughout the course of infection, P. aeruginosa must evolve and adapt from an initial state of early, transient colonization to chronic colonization of the airways. Here, we examined isolates of P. aeruginosa from children under the age of 3 years old with CF to determine genetic adaptations the bacterium undergoes during this early stage of colonization and infection. These isolates were collected when early aggressive antimicrobial therapy was not the standard of care and therefore highlight strain evolution under limited antibiotic pressure. Examination of specific phenotypic adaptations, such as lipid A palmitoylation, antibiotic resistance, and loss of quorum sensing, did not reveal a clear genetic basis for such changes. Additionally, we demonstrate that the geography of patient origin, within the United States or among other countries, does not appear to significantly influence genetic adaptation. In summary, our results support the long-standing model that patients acquire individual isolates of P. aeruginosa that subsequently become hyperadapted to the patient-specific airway environment. This study provides a multipatient genomic analysis of isolates from young CF patients in the United States and contributes data regarding early colonization and adaptation to the growing body of research about P. aeruginosa evolution in the context of CF airway disease. IMPORTANCE Chronic lung infection with Pseudomonas aeruginosa is of major concern for patients with cystic fibrosis (CF). During infection, P. aeruginosa undergoes genomic and functional adaptation to the hyperinflammatory CF airway, resulting in worsening lung function and pulmonary decline. All studies that describe these adaptations use P. aeruginosa obtained from older children or adults during late chronic lung infection; however, children with CF can be infected with P. aeruginosa as early as 3 months of age. Therefore, it is unclear when these genomic and functional adaptations occur over the course of CF lung infection, as access to P. aeruginosa isolates in children during early infection is limited. Here, we present a unique cohort of CF patients who were identified as being infected with P. aeruginosa at an early age prior to aggressive antibiotic therapy. Furthermore, we performed genomic and functional characterization of these isolates to address whether chronic CF P. aeruginosa phenotypes are present during early infection.

Ketogenesis promotes tolerance to Pseudomonas aeruginosa pulmonary infection.

Pseudomonas aeruginosa is a common cause of pulmonary infection. As a Gram-negative pathogen, it can initiate a brisk and highly destructive inflammatory response; however, most hosts become tolerant to the bacterial burden, developing chronic infection. Using a murine model of pneumonia, we demonstrate that this shift from inflammation to disease tolerance is promoted by ketogenesis. In response to pulmonary infection, ketone bodies are generated in the liver and circulate to the lungs where they impose selection for P. aeruginosa strains unable to display surface lipopolysaccharide (LPS). Such keto-adapted LPS strains fail to activate glycolysis and tissue-damaging cytokines and, instead, facilitate mitochondrial catabolism of fats and oxidative phosphorylation (OXPHOS), which maintains airway homeostasis. Within the lung, P. aeruginosa exploits the host immunometabolite itaconate to further stimulate ketogenesis. This environment enables host-P. aeruginosa coexistence, supporting both pathoadaptive changes in the bacteria and the maintenance of respiratory integrity via OXPHOS.