BECC-engineered live-attenuated Shigella vaccine candidates display reduced endotoxicity with robust immunogenicity in mice.

Shigella spp. infection contributes significantly to the global disease burden, primarily affecting young children in developing countries. Currently, there are no FDA-approved vaccines against Shigella, and the prevalence of antibiotic resistance is increasing, making therapeutic options limited. Live-attenuated vaccine strains WRSs2 (S. sonnei) and WRSf2G12 (S. flexneri 2a) are highly immunogenic, making them promising vaccine candidates, but possess an inflammatory lipid A structure on their lipopolysaccharide (LPS; also known as endotoxin). Here, we utilized bacterial enzymatic combinatorial chemistry (BECC) to ectopically express lipid A modifying enzymes in WRSs2 and WRSf2G12, as well as their respective wild-type strains, generating targeted lipid A modifications across the Shigella backgrounds. Dephosphorylation of lipid A, rather than deacylation, reduced LPS-induced TLR4 signaling in vitro and dampened endotoxic effects in vivo. These BECC-modified vaccine strains retained the phenotypic traits of their parental strains, such as invasion of epithelial cells and immunogenicity in mice without adverse endotoxicity. Overall, our observations suggest that BECC-engineered live attenuated vaccines are a promising approach to safe and effective Shigella vaccines.

The multifaceted role of c-di-AMP signaling in the regulation of Porphyromonas gingivalis lipopolysaccharide structure and function.

Background: This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. Methods: We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLATn. Results: We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. Conclusion: The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

Vaccination with a Protective Ipa Protein-Containing Nanoemulsion Differentially Alters the Transcriptomic Profiles of Young and Elderly Mice following Shigella Infection.

Shigella spp. are responsible for bacillary dysentery or shigellosis transmitted via the fecal-oral route, causing significant morbidity and mortality, especially among vulnerable populations. There are currently no licensed Shigella vaccines. Shigella spp. use a type III secretion system (T3SS) to invade host cells. We have shown that L-DBF, a recombinant fusion of the T3SS needle tip (IpaD) and translocator (IpaB) proteins with the LTA1 subunit of enterotoxigenic E. coli labile toxin, is broadly protective against Shigella spp. challenge in a mouse lethal pulmonary model. Here, we assessed the effect of LDBF, formulated with a unique TLR4 agonist called BECC470 in an oil-in-water emulsion (ME), on the murine immune response in a high-risk population (young and elderly) in response to Shigella challenge. Dual RNA Sequencing captured the transcriptome during Shigella infection in vaccinated and unvaccinated mice. Both age groups were protected by the L-DBF formulation, while younger vaccinated mice exhibited more adaptive immune response gene patterns. This preliminary study provides a step toward identifying the gene expression patterns and regulatory pathways responsible for a protective immune response against Shigella. Furthermore, this study provides a measure of the challenges that need to be addressed when immunizing an aging population.

Lipid A modification of colistin-resistant Klebsiella pneumoniae does not alter innate immune response in a mouse model of pneumonia.

Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.

Development of a nano-emulsion based multivalent protein subunit vaccine against Pseudomonas aeruginosa.

Pseudomonas aeruginosa (Pa) is an opportunistic bacterial pathogen responsible for severe hospital acquired infections in immunocompromised and elderly individuals. Emergence of increasingly drug resistant strains and the absence of a broad-spectrum prophylactic vaccine against both T3SA+ (type III secretion apparatus) and ExlA+/T3SA- Pa strains worsen the situation in a post-pandemic world. Thus, we formulated a candidate subunit vaccine (called ExlA/L-PaF/BECC/ME) against both Pa types. This bivalent vaccine was generated by combining the C-terminal active moiety of exolysin A (ExlA) produced by non-T3SA Pa strains with our T3SA-based vaccine platform, L-PaF, in an oil-in-water emulsion. The ExlA/L-PaF in ME (MedImmune emulsion) was then mixed with BECC438b, an engineered lipid A analogue and a TLR4 agonist. This formulation was administered intranasally (IN) to young and elderly mice to determine its potency across a diverse age-range. The elderly mice were used to mimic the infection seen in elderly humans, who are more susceptible to serious Pa disease compared to their young adult counterparts. After Pa infection, mice immunized with ExlA/L-PaF/BECC/ME displayed a T cell-mediated adaptive response while PBS-vaccinated mice experienced a rapid onset inflammatory response. Important genes and pathways were observed, which give rise to an anti-Pa immune response. Thus, this vaccine has the potential to protect aged individuals in our population from serious Pa infection.

Detection of a Human Adenovirus Outbreak, Including Some Critical Infections, Using Multipathogen Testing at a Large University, September 2022-January 2023.

Background: Human adenoviruses (HAdVs) can cause outbreaks of flu-like illness in university settings. Most infections in healthy young adults are mild; severe illnesses rarely occur. In Fall 2022, an adenovirus outbreak was identified in university students. Methods: HAdV cases were defined as university students 17-26 years old who presented to the University Health Service or nearby emergency department with flu-like symptoms (eg, fever, cough, headache, myalgia, nausea) and had confirmed adenovirus infections by polymerase chain reaction (PCR). Demographic and clinical characteristics were abstracted from electronic medical records; clinical severity was categorized as mild, moderate, severe, or critical. We performed contact investigations among critical cases. A subset of specimens was sequenced to confirm the HAdV type. Results: From 28 September 2022 to 30 January 2023, 90 PCR-confirmed cases were identified (51% female; mean age, 19.6 years). Most cases (88.9%) had mild illness. Seven cases required hospitalization, including 2 critical cases that required intensive care. Contact investigation identified 44 close contacts; 6 (14%) were confirmed HAdV cases and 8 (18%) reported symptoms but never sought care. All typed HAdV-positive specimens (n = 36) were type 4. Conclusions: While most students with confirmed HAdV had mild illness, 7 otherwise healthy students had severe or critical illness. Between the relatively high number of hospitalizations and proportion of close contacts with symptoms who did not seek care, the true number of HAdV cases was likely higher. Our findings illustrate the need to consider a wide range of pathogens, even when other viruses are known to be circulating.

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, not seen in any other disease state. Lipid A, the membrane anchor of lipopolysaccharide (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent toll-like receptor (TLR)4 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 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 bronchoalveolar lavage fluid. This structure triggers increased pro-inflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CFTR function. Interestingly, 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 lack 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.

Identification of a 2-Aminobenzimidazole Scaffold that Potentiates Gram-Positive Selective Antibiotics Against Gram-Negative Bacteria.

The development of novel therapeutic approaches is crucial in the fight against multi-drug resistant (MDR) bacteria, particularly gram-negative species. Small molecule adjuvants that enhance the activity of otherwise gram-positive selective antibiotics against gram-negative bacteria have the potential to expand current treatment options. We have previously reported adjuvants based upon a 2-aminoimidazole (2-AI) scaffold that potentiate macrolide antibiotics against several gram-negative pathogens. Herein, we report the discovery and structure-activity relationship (SAR) investigation of an additional class of macrolide adjuvants based upon a 2-aminobenzimidazole (2-ABI) scaffold. The lead compound lowers the minimum inhibitory concentration (MIC) of clarithromycin (CLR) from 512 to 2 µg/mL at 30 µM against Klebsiella pneumoniae 2146, and from 32 to 2 µg/mL at 5 µM, against Acinetobacter baumannii 5075. Preliminary investigation into the mechanism of action suggests that the compounds are binding to lipopolysaccharide (LPS) in K. pneumoniae, and modulating lipooligosaccharide (LOS) biosynthesis, assembly, or transport in A. baumannii.

MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress.

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.

Polymer-extracted structure of the mechanosensitive channel MscS reveals the role of protein-lipid interactions in the gating cycle.

Membrane protein structure determination is not only technically challenging but is further complicated by the removal or displacement of lipids, which can result in non-native conformations or a strong preference for certain states at the exclusion of others. This is especially applicable to mechanosensitive channels (MSC's) that evolved to gate in response to subtle changes in membrane tension transmitted through the lipid bilayer. E. coli MscS, a model bacterial system, is an ancestral member of the large family of MSCs found across all phyla of walled organisms. As a tension sensor, MscS is very sensitive and highly adaptive; it readily opens under super-threshold tension and closes under no tension, but under lower tensions, it slowly inactivates and can only recover when tension is released. However, existing cryo-EM structures do not explain the entire functional gating cycle of open, closed, and inactivated states. A central question in the field has been the assignment of the frequently observed non-conductive conformation to either a closed or inactivated state. Here, we present a 3 Å MscS structure in native nanodiscs obtained with Glyco-DIBMA polymer extraction, eliminating the lipid removal step that is common to all previous structures. Besides the protein in the non-conductive conformation, we observe well-resolved densities of four endogenous phospholipid molecules intercalating between the lipid-facing and pore-lining helices in preferred orientations. Mutations of positively charged residues coordinating these lipids inhibit MscS inactivation, whereas removal of a negative charge near the lipid-filled crevice increases inactivation. The functional data allows us to assign this class of structures to the inactivated state. This structure reveals preserved lipids in their native locations, and the functional effects of their destabilization illustrate a novel inactivation mechanism based on an uncoupling of the peripheral tension-sensing helices from the gate.

BECC438b TLR4 agonist supports unique immune response profiles from nasal and muscular DTaP pertussis vaccines in murine challenge models.

The protection afforded by acellular pertussis vaccines wanes over time, and there is a need to develop improved vaccine formulations. Options to improve the vaccines involve the utilization of different adjuvants and administration via different routes. While intramuscular (IM) vaccination provides a robust systemic immune response, intranasal (IN) vaccination theoretically induces a localized immune response within the nasal cavity. In the case of a Bordetella pertussis infection, IN vaccination results in an immune response that is similar to natural infection, which provides the longest duration of protection. Current acellular formulations utilize an alum adjuvant, and antibody levels wane over time. To overcome the current limitations with the acellular vaccine, we incorporated a novel TLR4 agonist, BECC438b, into both IM and IN acellular formulations to determine its ability to protect against infection in a murine airway challenge model. Following immunization and challenge, we observed that DTaP + BECC438b reduced bacterial burden within the lung and trachea for both administration routes when compared with mock-vaccinated and challenged (MVC) mice. Interestingly, IN administration of DTaP + BECC438b induced a Th1-polarized immune response, while IM vaccination polarized toward a Th2 immune response. RNA sequencing analysis of the lung demonstrated that DTaP + BECC438b activates biological pathways similar to natural infection. Additionally, IN administration of DTaP + BECC438b activated the expression of genes involved in a multitude of pathways associated with the immune system. Overall, these data suggest that BECC438b adjuvant and the IN vaccination route can impact efficacy and responses of pertussis vaccines in pre-clinical mouse models.

Structural determination of Rickettsia lipid A without chemical extraction confirms shorter acyl chains in later-evolving spotted fever group pathogens.

Rickettsiae are Gram-negative obligate intracellular parasites of numerous eukaryotes. Human pathogens of the transitional group (TRG), typhus group (TG), and spotted fever group (SFG) rickettsiae infect blood-feeding arthropods, have dissimilar clinical manifestations, and possess unique genomic and morphological attributes. Lacking glycolysis, rickettsiae pilfer numerous metabolites from the host cytosol to synthesize peptidoglycan and lipopolysaccharide (LPS). For LPS, O-antigen immunogenicity varies between SFG and TG pathogens; however, lipid A proinflammatory potential is unknown. We previously demonstrated that Rickettsia akari (TRG), Rickettsia typhi (TG), and Rickettsia montanensis (SFG) produce lipid A with long 2' secondary acyl chains (C16 or C18) compared to short 2' secondary acyl chains (C12) in Rickettsia rickettsii (SFG) lipid A. To further probe this structural heterogeneity and estimate a time point when shorter 2' secondary acyl chains originated, we generated lipid A structures for two additional SFG rickettsiae (Rickettsia rhipicephali and Rickettsia parkeri) utilizing fast lipid analysis technique adopted for use with tandem mass spectrometry (FLATn). FLATn allowed analysis of lipid A structure directly from host cell-purified bacteria, providing a substantial improvement over lipid A chemical extraction. FLATn-derived structures indicate SFG rickettsiae diverging after R. rhipicephali evolved shorter 2' secondary acyl chains. While 2' secondary acyl chain lengths do not distinguish Rickettsia pathogens from non-pathogens, in silico analyses of Rickettsia LpxL late acyltransferases revealed discrete active sites and hydrocarbon rulers for long versus short 2' secondary acyl chain addition. Our collective data warrant determining Rickettsia lipid A inflammatory potential and how structural heterogeneity impacts lipid A-host receptor interactions.IMPORTANCEDeforestation, urbanization, and homelessness lead to spikes in Rickettsioses. Vector-borne human pathogens of transitional group (TRG), typhus group (TG), and spotted fever group (SFG) rickettsiae differ by clinical manifestations, immunopathology, genome composition, and morphology. We previously showed that lipid A (or endotoxin), the membrane anchor of Gram-negative bacterial lipopolysaccharide (LPS), structurally differs in Rickettsia rickettsii (later-evolving SFG) relative to Rickettsia montanensis (basal SFG), Rickettsia typhi (TG), and Rickettsia akari (TRG). As lipid A structure influences recognition potential in vertebrate LPS sensors, further assessment of Rickettsia lipid A structural heterogeneity is needed. Here, we sidestepped the difficulty of ex vivo lipid A chemical extraction by utilizing fast lipid analysis technique adopted for use with tandem mass spectrometry, a new procedure for generating lipid A structures directly from host cell-purified bacteria. These data confirm that later-evolving SFG pathogens synthesize structurally distinct lipid A. Our findings impact interpreting immune responses to different Rickettsia pathogens and utilizing lipid A adjuvant or anti-inflammatory properties in vaccinology.

Endoplasmic Reticulum Membrane Homeostasis and the Unfolded Protein Response.

The endoplasmic reticulum (ER) is the key organelle for membrane biogenesis. Most lipids are synthesized in the ER, and most membrane proteins are first inserted into the ER membrane before they are transported to their target organelle. The composition and properties of the ER membrane must be carefully controlled to provide a suitable environment for the insertion and folding of membrane proteins. The unfolded protein response (UPR) is a powerful signaling pathway that balances protein and lipid production in the ER. Here, we summarize our current knowledge of how aberrant compositions of the ER membrane, referred to as lipid bilayer stress, trigger the UPR.

Recombinant Interleukin-1 Receptor Antagonist Is an Effective First-Line Treatment Strategy in New-Onset Systemic Juvenile Idiopathic Arthritis, Irrespective of HLA-DRB1 Background and IL1RN Variants.

OBJECTIVE: Human leukocyte antigen (HLA)-DRB1*15:01 has been recently associated with interstitial lung disease (LD), eosinophilia, and drug reactions in systemic juvenile idiopathic arthritis (sJIA). Additionally, genetic variants in IL1RN have been linked to poor response to anakinra. We sought to reproduce these findings in a prospective cohort study of patients with new-onset sJIA treated with anakinra as first-line therapy. METHODS: HLA and IL1RN risk alleles were identified via whole-genome sequencing. Treatment responses and complications were compared between carriers versus noncarriers. RESULTS: Seventeen of 65 patients (26%) carried HLA-DRB1*15:01, comparable with the general population, and there was enrichment for HLA-DRB1*11:01, a known risk locus for sJIA. The rates of clinical inactive disease (CID) at 6 months, 1 year, and 2 years were generally high, irrespective of HLA-DRB1 or IL1RN variants, but significantly lower in carriers of an HLA-DRB1*11:01 allele. One patient, an HLA-DRB1*15:01 carrier, developed sJIA-LD. Of the three patients with severe drug reactions to biologics, one carried HLA-DRB1*15:01. The prevalence of eosinophilia did not significantly differ between HLA-DRB1*15:01 carriers and noncarriers at disease onset (6.2% vs 14.9%, P = 0.67) nor after the start of anakinra (35.3% vs 37.5% in the first 2 years of disease). CONCLUSION: We observed high rates of CID using anakinra as first-line treatment irrespective of HLA-DRB1 or IL1RN variants. Only one of the 17 HLA-DRB1*15:01 carriers developed sJIA-LD, and of the three patients with drug reactions to biologics, only one carried HLA-DRB1*15:01. Although thorough monitoring for the development of drug hypersensitivity and refractory disease courses in sJIA, including sJIA-LD, remains important, our data support the early start of biologic therapy in patients with new-onset sJIA irrespective of HLA-DRB1 background or IL1RN variants.

Comparison of three rapid diagnostic tests for bloodstream infections using Benefit-risk Evaluation Framework (BED-FRAME).

Rapid diagnostic tests (RDTs) for bloodstream infections have the potential to reduce time to appropriate antimicrobial therapy and improve patient outcomes. Previously, an in-house, lipid-based, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) method, Fast Lipid Analysis Technique (FLAT MS), has shown promise as a rapid pathogen identification method. In this study, FLAT MS for direct from blood culture identification was evaluated and compared to FDA-cleared identification methods using the Benefit-risk Evaluation Framework (BED-FRAME) analysis. FLAT MS was evaluated and compared to Bruker Sepsityper and bioMérieux BioFire FilmArray BCID2 using results from a previous study. For this study, 301 positive blood cultures were collected from the University of Maryland Medical Center. The RDTs were compared by their sensitivities, time-to-results, hands-on time, and BED-FRAME analysis. The overall sensitivity of all platforms compared to culture results from monomicrobial-positive blood cultures was 88.3%. However, the three RDTs differed in their accuracy for identifying Gram-positive bacteria, Gram-negative bacteria, and yeast. Time-to-results for FLAT MS, Sepsityper, and BioFire BCID2 were all approximately one hour. Hands-on times for FLAT MS, Sepsityper, and BioFire BCID2 were 10 (±1.3), 40 (±2.8), and 5 (±0.25) minutes, respectively. BED-FRAME demonstrated that each RDT had utility at different pathogen prevalence and relative importance. BED-FRAME is a useful tool that can used to determine which RDT is best for a healthcare center.

Sex differences in visceral sensitivity and brain activity in a rat model of comorbid pain: a longitudinal study.

ABSTRACT: Temporomandibular disorder (TMD) and irritable bowel syndrome (IBS) are 2 chronic overlapping pain conditions (COPCs) that present with significant comorbidity. Both conditions are more prevalent in women and are exacerbated by stress. While peripheral mechanisms might contribute to pain hypersensitivity for each individual condition, mechanisms underlying the comorbidity are poorly understood, complicating pain management when multiple conditions are involved. In this study, longitudinal behavioral and functional MRI-based brain changes have been identified in an animal model of TMD-like pain (masseter muscle inflammation followed by stress) that induces de novo IBS-like comorbid visceral pain hypersensitivity in rats. In particular, data indicate that increased activity in the insula and regions of the reward and limbic systems are associated with more pronounced and longer-lasting visceral pain behaviors in female rats, while the faster pain resolution in male rats may be due to increased activity in descending pain inhibitory pathways. These findings suggest the critical role of brain mechanisms in chronic pain conditions and that sex may be a risk factor of developing COPCs.

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.

Lack of Evidence for the Role of the p.(Ser96Ala) Polymorphism in Histidine-Rich Calcium Binding Protein as a Secondary Hit in Cardiomyopathies.

Inherited forms of arrhythmogenic and dilated cardiomyopathy (ACM and DCM) are characterized by variable disease expression and age-related penetrance. Calcium (Ca2+) is crucially important for proper cardiac function, and dysregulation of Ca2+ homeostasis seems to underly cardiomyopathy etiology. A polymorphism, c.286T>G p.(Ser96Ala), in the gene encoding the histidine-rich Ca2+ binding (HRC) protein, relevant for sarcoplasmic reticulum Ca2+ cycling, has previously been associated with a marked increased risk of life-threatening arrhythmias among idiopathic DCM patients. Following this finding, we investigated whether p.(Ser96Ala) affects major cardiac disease manifestations in carriers of the phospholamban (PLN) c.40_42delAGA; p.(Arg14del) pathogenic variant (cohort 1); patients diagnosed with, or predisposed to, ACM (cohort 2); and DCM patients (cohort 3). We found that the allele frequency of the p.(Ser96Ala) polymorphism was similar across the general European-American population (control cohort, 40.3-42.2%) and the different cardiomyopathy cohorts (cohorts 1-3, 40.9-43.9%). Furthermore, the p.(Ser96Ala) polymorphism was not associated with life-threatening arrhythmias or heart failure-related events across various patient cohorts. We therefore conclude that there is a lack of evidence supporting the important role of the HRC p.(Ser96Ala) polymorphism as a modifier in cardiomyopathy, refuting previous findings. Further research is required to identify bona fide genomic predictors for the stratification of cardiomyopathy patients and their risk for life-threatening outcomes.