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Purpose: Quality improvement (QI) processes provide a framework for systematically examining target outcomes and what changes can be made to result in improvement and ensure equity. We present a case study of how QI processes were used as a means of partnership building to enhance equity in designing materials for a Medicaid pilot program, North Carolina Integrated Care for Kids (NC InCK)., Description: The NC InCK model addresses social determinants of health by providing structured care integration across core child health and social service areas and using an alternative payment model to incentivize high quality child outcomes. During the two-year planning period prior to the NC InCK model launch, we used Plan-Do-Study-Act (PDSA) cycles to conduct usability testing as a QI strategy for a component of the NC InCK model: the Shared Action Plan (SAP)., Assessment: We conducted usability testing with four Family Council members, nine care managers, and one physician. Participants reviewed the SAP and provided feedback via a survey. After reviewing feedback with InCK leadership and the Family Council, we implemented recommendations that led to a SAP that uses clear and accessible language, that highlights family strengths and family-identified goals, and that is distinct from other care management plans., Conclusion: Usability testing forced refinement of materials before NC InCK launched, created opportunities for building and enhancing community partnerships and promoted equity within the NC InCK team and Family Council by considering multiple perspectives when deciding on SAP revisions., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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 (FLAT n ). FLAT n allowed analysis of lipid A structure directly from host cell-purified bacteria, providing a substantial improvement over lipid A chemical extraction. FLAT n -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., Competing Interests: The authors declare no conflict of interest.

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.IMPORTANCE Pseudomonas 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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no conflict of interest.

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 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 R. akari (TRG), R. typhi (TG), and R. montanensis (SFG) produce lipid A with long 2' secondary acyl chains (C16 or C18) compared to short 2' secondary acyl chains (C12) in R. 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 ( R. rhipicephali and R. parkeri ) utilizing Fast Lipid Analysis Technique adopted for use with tandem mass spectrometry (FLAT n ). FLAT n allowed analysis of lipid A structure directly from host cell-purified bacteria, providing substantial improvement over lipid A chemical extraction. FLAT n -derived structures indicate SFG rickettsiae diverging after R. rhipicephali evolved shorter 2' secondary acyl chains. Bioinformatics analysis of Rickettsia LpxL late acyltransferases revealed discrete active sites and hydrocarbon rulers for long versus short 2' secondary acyl chain addition. While the significance of different lipid A structures for diverse Rickettsia pathogens is unknown, our success using FLAT n will facilitate determining how structural heterogeneity impacts interactions with host lipid A receptors and overall inflammatory potential., Importance: Deforestation, 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 R. rickettsii (later-evolving SFG) relative to R. montanensis (basal SFG), R. typhi (TG), and R. 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 FLAT n , a new procedure for generating lipid A structures directly from host cell-purified bacteria. These data confirm 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.

Quantitative proteomics has matured into an established tool and longitudinal proteomics experiments have begun to emerge. However, no effective, simple-to-use differential expression method for longitudinal proteomics data has been released. Typically, such data is noisy, contains missing values, and has only few time points and biological replicates. To address this need, we provide a comprehensive evaluation of several existing differential expression methods for high-throughput longitudinal omics data and introduce a Robust longitudinal Differential Expression (RolDE) approach. The methods are evaluated using over 3000 semi-simulated spike-in proteomics datasets and three large experimental datasets. In the comparisons, RolDE performs overall best; it is most tolerant to missing values, displays good reproducibility and is the top method in ranking the results in a biologically meaningful way. Furthermore, RolDE is suitable for different types of data with typically unknown patterns in longitudinal expression and can be applied by non-experienced users., (© 2022. The Author(s).)

Whole cell vaccines are complex mixtures of antigens, immunogens, and sometimes adjuvants that can trigger potent and protective immune responses. In some instances, such as whole cell Bordetella pertussis vaccination, the immune response to vaccination extends beyond the pathogen the vaccine was intended for and contributes to protection against other clinically significant pathogens. In this study, we describe how B. pertussis whole cell vaccination protects mice against acute pneumonia caused by Pseudomonas aeruginosa. Using ELISA and western blot, we identified that B. pertussis whole cell vaccination induces production of antibodies that bind to lab-adapted and clinical strains of P. aeruginosa, regardless of immunization route or adjuvant used. The cross-reactive antigens were identified using immunoprecipitation, mass spectrometry, and subsequent immunoblotting. We determined that B. pertussis GroEL and OmpA present in the B. pertussis whole cell vaccine led to production of antibodies against P. aeruginosa GroEL and OprF, respectively. Finally, we showed that recombinant B. pertussis OmpA was sufficient to induce protection against P. aeruginosa acute murine pneumonia. This study highlights the potential for use of B. pertussis OmpA as a vaccine antigen for prevention of P. aeruginosa infection, and the potential of broadly protective antigens for vaccine development., (© 2022. The Author(s).)

Kingella kingae is a leading cause of bone and joint infections and other invasive diseases in young children. A key K. kingae virulence determinant is a secreted exopolysaccharide that mediates resistance to serum complement and neutrophils and is required for full pathogenicity. The K. kingae exopolysaccharide is a galactofuranose homopolymer called galactan and is encoded by the pamABC genes in the pamABCDE locus. In this study, we sought to define the mechanism by which galactan is tethered on the bacterial surface, a prerequisite for mediating evasion of host immune mechanisms. We found that the pamD and pamE genes encode glycosyltransferases and are required for synthesis of an atypical lipopolysaccharide (LPS) O-antigen. The LPS O-antigen in turn is required for anchoring of galactan, a novel mechanism for association of an exopolysaccharide with the bacterial surface. IMPORTANCE Kingella kingae is an emerging pediatric pathogen and produces invasive disease by colonizing the oropharynx, invading the bloodstream, and disseminating to distant sites. This organism produces a uniquely multifunctional exopolysaccharide called galactan that is critical for virulence and promotes intravascular survival by mediating resistance to serum and neutrophils. In this study, we established that at least some galactan is anchored to the bacterial surface via a novel structural interaction with an atypical lipopolysaccharide O-antigen. Additionally, we demonstrated that the atypical O-antigen is synthesized by the products of the pamD and pamE genes, located downstream of the gene cluster responsible for galactan biosynthesis. This work addresses how the K. kingae exopolysaccharide can mediate innate immune resistance and advances understanding of bacterial exopolysaccharides and lipopolysaccharides.

We describe an innovative use for the recently reported fast lipid analysis technique (FLAT) that allows for the generation of MALDI tandem mass spectrometry data suitable for lipid A structure analysis directly from a single Gram-negative bacterial colony. We refer to this tandem MS version of FLAT as FLAT n . Neither technique requires sophisticated sample preparation beyond the selection of a single bacterial colony, which significantly reduces overall analysis time (∼1 h), as compared to conventional methods. Moreover, the tandem mass spectra generated by FLAT n provides comprehensive information on fragments of lipid A, for example, ester bonded acyl chain dissociations, cross-ring cleavages, and glycosidic bond dissociations, all of which allow the facile determination of novel lipid A structures or confirmation of expected structures. In addition to generating tandem mass spectra directly from single colonies, we also show that FLAT n can be used to analyze lipid A structures taken directly from a complex biological clinical sample without the need for ex vivo growth. From a urine sample from a patient with an E. coli infection, FLAT n identified the organism and demonstrated that this clinical isolate carried the mobile colistin resistance-1 gene ( mcr-1 ) that results in the addition of a phosphoethanolamine moiety and subsequently resistance to the antimicrobial, colistin (polymyxin E). Moreover, FLAT n allowed for the determination of the existence of a structural isomer in E. coli lipid A that had either a 1- or 4'-phosphate group modification by phosphoethanolamine generated by a change of bacterial culture conditions.

Child maltreatment is a significant public health issue in the United States. Understanding key risk factors for child maltreatment is critical to informing effective prevention. Poverty is an established risk factor for child maltreatment. However, recent research indicates that material hardship (i.e., difficulties meeting basic needs) may serve as a more direct measure of the way in which poverty affects daily life. One form of material hardship that is common among families is housing stress. Previous reviews have summarized the existing literature regarding the association of economic insecurity with child maltreatment, but no reviews have synthesized and critically evaluated the literature specific to the association of various types of housing stress with child maltreatment. We conducted a systematic search of multiple electronic databases to identify peer-reviewed studies conducted in the U.S. regarding the association of housing stress with child maltreatment. We identified 21 articles that used nine distinct measures of housing stress including homelessness or eviction, homeless or emergency shelter stays, foreclosure filing, housing instability, inadequate housing, physical housing risk, living doubled-up, housing unaffordability, and composite housing stress indicators. Overall, results from this body of literature indicate that housing stress is associated with an increased likelihood of caregiver or child self-reported maltreatment, child protective services (CPS) reports, investigated and substantiated CPS reports, out-of-home placements, and maltreatment death. Additional theory-driven research is needed to further our understanding of the contribution of specific types of housing stress to risk for specific types of maltreatment.

Candida auris is an emerging multidrug-resistant fungal pathogen. With high mortality rates, there is an urgent need for new antifungals to combat C. auris . Possible antifungal targets include Cu-only superoxide dismutases (SODs), extracellular SODs that are unique to fungi and effectively combat the superoxide burst of host immunity. Cu-only SODs are essential for the virulence of diverse fungal pathogens; however, little is understood about these enzymes in C. auris . We show here that C. auris secretes an enzymatically active Cu-only SOD ( Caur SOD4) when cells are starved for Fe, a condition mimicking host environments. Although predicted to attach to cell walls, Caur SOD4 is detected as a soluble extracellular enzyme and can act at a distance to remove superoxide. Caur SOD4 selectively binds Cu and not Zn, and Cu binding is labile compared to bimetallic Cu/Zn SODs. Moreover, Caur SOD4 is susceptible to inhibition by various metal-binding drugs that are without effect on mammalian Cu/Zn SODs. Our studies highlight Caur SOD4 as a potential antifungal target worthy of consideration.

In an attempt to move the field of public health from documenting health disparities to acting to rectify them, in 2001, the American Public Health Association (APHA) recognized racism as a fundamental cause of racial health disparities. Both APHA and the Council on Education for Public Health have moved to incorporate new competencies in health equity for public health professionals. As schools and programs of public health work to establish curricular offerings in race and racism, a need exists to identify approaches currently in use that can be replicated, adapted, and scaled. This systematic review sought to identify pedagogical methods and curricula that exist to support the training of US public health students in understanding racism as a structural determinant of health. We found 11 examples from peer-reviewed literature of curricula, lessons, and competencies that have been developed by public health faculty and departments since 2006. The articles discussed a range of approaches to teaching about structural racism in public health, suggesting that little consensus may exist on how to best teach this material. Furthermore, we found little rigorous evaluation of these teaching methods and curricula. The results of this review suggest future research is needed on public health pedagogy on structural racism.

Enterobacter species are classified as high-priority pathogens due to high prevalence of multidrug resistance from persistent antibiotic use. For Enterobacter infections caused by multidrug-resistant isolates, colistin (polymyxin E), a last-resort antibiotic, is a potential treatment option. Treatment with colistin has been shown to lead to emergence of polymyxin resistance. The primary mechanism for colistin resistance is modification of terminal phosphate moieties of lipid A, leading to decreased membrane electronegativity and reducing colistin binding affinity. Detection of these modifications, including the addition of phosphoethanolamine and 4-amino-4-deoxy-l-arabinose (Ara4N), can be used for prediction of colistin resistance using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The objective of this study was to identify lipid A markers for colistin resistance in Enterobacter species and Klebsiella aerogenes (formerly Enterobacter aerogenes). Using a collection of Enterobacter and Klebsiella aerogenes clinical isolates, broth MICs for colistin were determined initially. Subsequently, killing assays were carried out to determine how the concentration of colistin at which there is approximately 50% survival (kill 50 ) equates to their MICs. Finally, lipid A analysis was conducted via MALDI-TOF MS using the novel rapid extraction method, termed fast lipid analysis technique (FLAT), to correlate MIC and killing efficacy with predictive lipid A modifications. Sensitivity and specificity of the MS assay compared to MIC interpretation were 100% and 53.4%, respectively. A receiver operator characteristic (ROC) demonstrated that MS was highly correlated with killing, with area under the curve of 0.97. This analysis demonstrated the potential utility of MALDI-TOF MS as a rapid diagnostic platform of colistin resistance in Enterobacter species. IMPORTANCE In this study, we develop a novel method for identifying colistin resistance in Enterobacter species and Klebsiella aerogenes without performing antimicrobial susceptibility testing. Typically, susceptibility testing requires an additional 24 to 48 h, while the MS assay described in this study allows for resistant identifications in under 1 h after initial culture. Identification using MALDI-TOF MS would save time and prevent inappropriate use of colistin. MALDI-TOF MS is an easy-to-use, readily available, robust diagnostic tool in clinical laboratories. Furthermore, this study highlights limitations of polymyxin susceptibility testing. Use of a killing assay best captures how colistin treats infection and is shown to be highly correlated with our MS assay; thus, the MS assay in this study effectively predicts how colistin would treat a patient's infection. Use of MALDI-TOF MS for accurate and early identification of antimicrobial resistance can improve antimicrobial stewardship and patient outcomes.

The third annual conference of the Imaging Mass Spectrometry Society (IMSS3) was held October 3-6, 2021 in a hybrid format that included virtual and in-person attendance (Colorado Springs, CO). Here, we highlight many of the methods and applications presented, the state of the field, and some insights into the emerging areas in the field of imaging mass spectrometry. We also reflect upon the processes behind planning a hybrid conference and discuss the successes and challenges of the event in retrospect.

Background: Longitudinal studies on resilience among children who have experienced maltreatment indicate that resilience is multi-dimensional. However, most research consolidates diverse developmental domains comprising resilience into a single score, which does not allow for detection of potentially heterogeneous associations between risk factors and outcomes of resilience processes., Objective: This study seeks to improve our understanding of the association between early child maltreatment and development through middle childhood (6-12 years) using individual domains considered to be outcomes of resilience processes., Participants and Setting: Participants are 499 children from the Longitudinal Studies of Child Abuse and Neglect., Methods: We used latent growth curve models to explore patterns of socialization and daily living skills, and internalizing and externalizing behaviors - outcomes of resilience processes - across three time points in middle childhood, and their association with early maltreatment, defined as referral to Child Protective Services (CPS) before age 6., Results: In fully adjusted models, children experiencing early maltreatment had poorer baseline scores in activities of daily living (-4.22, 95% CI [-7.38, -1.46]) and externalizing behavior (2.95, 95% CI [1.05, 4.86]), but maltreatment was not associated with change over time in these domains. However, maltreatment was associated with increases in internalizing behavior over time (0.42, 95% CI [0.06, 0.77])., Conclusion: Heterogeneity in patterns of association between maltreatment and outcomes of resilience processes support the utility of examining developmental domains individually, versus as a composite, to identify specific targets for intervention., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Transporters belonging to the r esistance- n odulation- d ivision (RND) superfamily of proteins are invariably present in the genomes of Gram-negative bacteria and are largely responsible for the intrinsic antibiotic resistance of these organisms. The numbers of genes encoding RND transporters per genome vary from 1 to 16 and correlate with the environmental versatilities of bacterial species. Pseudomonas aeruginosa strain PAO1, a ubiquitous nosocomial pathogen, possesses 12 RND pumps, which are implicated in the development of clinical multidrug resistance and known to contribute to virulence, quorum sensing, and many other physiological functions. In this study, we analyzed how P. aeruginosa's physiology adapts to a lack of RND-mediated efflux activities. A combination of transcriptomics, metabolomics, genetic, and analytical approaches showed that the P. aeruginosa PΔ6 strain, lacking the six best-characterized RND pumps, activates a specific adaptation response that involves significant changes in the abundance and activities of several transport system, quorum sensing, iron acquisition, and lipid A modification pathways. Our results demonstrate that these cells accumulate large quantities of Pseudomonas quinolone signals (PQS), which triggers iron starvation and activation of siderophore biosynthesis and acquisition pathways. The accumulation of iron in turn activates lipid A modification and membrane protection pathways. A transcriptionally regulated RND pump, MuxABC-OpmB, contributes to these transformations by controlling the concentration of coumarins. Our results suggest that these changes reduce the permeability barrier of the outer membrane and are needed to protect the cell envelope of efflux-deficient P. aeruginosa.

Species of Rickettsia ( Alphaproteobacteria : Rickettsiales ) are obligate intracellular parasites of a wide range of eukaryotes, with recognized arthropod-borne human pathogens belonging to the transitional group (TRG), typhus group (TG), and spotted fever group (SFG) rickettsiae. Growing in the host cytosol, rickettsiae pilfer numerous metabolites to make a typical Gram-negative bacterial cell envelope. The O-antigen of rickettsial lipopolysaccharide (LPS) is immunogenic and has been shown to tether the S-layer to the rickettsial surface; however, little is known about the structure and immunogenicity of the Rickettsia lipid A moiety. The structure of lipid A, the membrane anchor of LPS, affects the ability of this molecule to interact with components of the host innate immune system, specifically the MD-2/TLR4 receptor complex. To dissect the host responses that can occur during Rickettsia in vitro and in vivo infection, structural analysis of Rickettsia lipid A is needed. Lipid A was extracted from four Rickettsia species and structurally analyzed. R. akari (TRG), R. typhi (TG), and R. montanensis (SFG) produced a similar structure, whereas R. rickettsii (SFG) altered the length of a secondary acyl group. While all structures have longer acyl chains than known highly inflammatory hexa-acylated lipid A structures, the R. rickettsii modification should differentially alter interactions with the hydrophobic internal pocket in MD2. The significance of these characteristics toward inflammatory potential as well as membrane dynamics between arthropod and vertebrate cellular environments warrants further investigation. Our work adds lipid A to the secretome and O-antigen as variable factors possibly correlating with phenotypically diverse rickettsioses. IMPORTANCE Spikes in rickettsioses occur as deforestation, urbanization, and homelessness increase human exposure to blood-feeding arthropods. Still, effective Rickettsia vaccines remain elusive. Recent studies have determined that Rickettsia lipopolysaccharide anchors the protective S-layer to the bacterial surface and elicits bactericidal antibodies. Furthermore, growing immunological evidence suggests vertebrate sensors (MD-2/TLR4 and noncanonical inflammasome) typically triggered by the lipid A portion of lipopolysaccharide are activated during Rickettsia infection. However, the immunopotency of Rickettsia lipid A is unknown due to poor appreciation for its structure. We determined lipid A structures for four distinct rickettsiae, revealing longer acyl chains relative to highly inflammatory bacterial lipid A. Surprisingly, lipid A of the Rocky Mountain spotted fever agent deviates in structure from other rickettsiae. Thus, lipid A divergence may contribute to variable disease phenotypes, sounding an alarm for determining its immunopotency and possible utility (i.e., as an adjuvant or anti-inflammatory) for development of more prudent rickettsiacidal therapies., (Copyright © 2021 Guillotte et al.)

The assumption of near-universal bacterial detection by pattern recognition receptors is a foundation of immunology. The limits of this pattern recognition concept, however, remain undefined. As a test of this hypothesis, we determined whether mammalian cells can recognize bacteria that they have never had the natural opportunity to encounter. These bacteria were cultivated from the deep Pacific Ocean, where the genus Moritella was identified as a common constituent of the culturable microbiota. Most deep-sea bacteria contained cell wall lipopolysaccharide (LPS) structures that were expected to be immunostimulatory, and some deep-sea bacteria activated inflammatory responses from mammalian LPS receptors. However, LPS receptors were unable to detect 80% of deep-sea bacteria examined, with LPS acyl chain length being identified as a potential determinant of immunosilence. The inability of immune receptors to detect most bacteria from a different ecosystem suggests that pattern recognition strategies may be defined locally, not globally., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Rapid infection diagnosis is critical to improving patient treatment and outcome. Recent studies have shown microbial lipids to be sensitive and selective biomarkers for identifying bacterial and fungal species and antimicrobial resistance. Practical procedures for microbial lipid biomarker analysis will therefore improve patient outcomes and antimicrobial stewardship. However, current lipid extraction methods require significant hands-on time and are thus not suited for direct adoption as a clinical assay for microbial identification. Here, we have developed a method for lipid extraction directly on the surface of stainless-steel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) plates, termed fast lipid analysis technique or FLAT, which facilitates the identification of bacterial and fungal species using a sub-60-minute workflow. Additionally, our method detects lipid A modifications in Gram-negative bacteria that are associated with antimicrobial resistance, including to colistin.

We developed a method to directly detect and map the Gram-negative bacterial virulence factor lipid A derived from lipopolysaccharide (LPS) by coupling acid hydrolysis with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). As the structure of lipid A (endotoxin) determines the innate immune outcome during infection, the ability to map its location within an infected organ or animal is needed to understand localized inflammatory responses that results during host-pathogen interactions. We previously demonstrated detection of free lipid A from infected tissue; however detection of lipid A derived from intact (smooth) LPS from host-pathogen MSI studies, proved elusive. Here, we detected LPS-derived lipid A from the Gram-negative pathogens, Escherichia coli ( Ec , m / z 1797) and Pseudomonas aeruginosa ( Pa , m / z 1446) using on-tissue acid hydrolysis to cleave the glycosidic linkage between the polysaccharide (core and O-antigen) and lipid A moieties of LPS. Using accurate mass methods, the ion corresponding to the major Ec and Pa lipid A species ( m / z 1797 and 1446, respectively) were unambiguously discriminated from complex tissue substrates. Further, we evaluated potential delocalization and signal loss of other tissue lipids and found no evidence for either, making this LPS-to-Lipid A-MSI (LLA-MSI) method, compatible with simultaneous host-pathogen lipid imaging following acid hydrolysis. This spatially sensitive technique is the first step in mapping host-influenced de novo lipid A modifications, such as those associated with antimicrobial resistance phenotypes, during Gram-negative bacterial infection and will advance our understanding of the host-pathogen interface.

Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) species are opportunistic lung pathogens of cystic fibrosis (CF) patients. While P. aeruginosa can initiate long-term infections in younger CF patients, Bcc infections only arise in teenagers and adults. Both P. aeruginosa and Bcc use type VI secretion systems (T6SSs) to mediate interbacterial competition. Here, we show P. aeruginosa isolates from teenage and adult CF patients, but not those from young CF patients, are outcompeted by the epidemic Bcc isolate Burkholderia cenocepacia strain AU1054 in a T6SS-dependent manner. The genomes of susceptible P. aeruginosa isolates harbor T6SS-abrogating mutations, the repair of which, in some cases, rendered the isolates resistant. Moreover, seven of eight Bcc strains outcompeted P. aeruginosa strains isolated from the same patients. Our findings suggest certain mutations that arise as P. aeruginosa adapts to the CF lung abrogate T6SS activity, making P. aeruginosa and its human host susceptible to potentially fatal Bcc superinfection., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis ( Ypt ) and Yersinia enterocolitica ( Ye ), as well as the causative agent of plague, Yersinia pestis ( Yp ). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye , lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2' and 3' positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp , a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion., Competing Interests: The authors declare no competing interest.

Acinetobacter baumannii infects a wide range of anatomic sites including the respiratory tract and bloodstream. Despite its clinical importance, little is known about the molecular basis of A. baumannii pathogenesis. We previously identified the UDP-N-acetyl-d-galactosaminuronic acid (UDP-GalNAcA) biosynthesis genes, gna-gne2, as being critical for survival in vivo. Herein, we demonstrate that Gna-Gne2 are part of a complex network connecting in vivo fitness, cell envelope homeostasis and resistance to antibiotics. The ∆gna-gne2 mutant exhibits a severe fitness defect during bloodstream infection. Capsule production is abolished in the mutant strain, which is concomitant with its inability to survive in human serum. In addition, the ∆gna-gne2 mutant was more susceptible to vancomycin and unable to grow on MacConkey plates, indicating an alteration in cell envelope integrity. Analysis of lipid A by mass spectrometry showed that the hexa- and hepta-acylated species were affected in the gna-gne2 mutant. Finally, the ∆gna-gne2 mutant was more susceptible to several classes of antibiotics. Together, this study demonstrates the importance of UDP-GalNAcA in the pathobiology of A. baumannii. By interrupting its biosynthesis, we showed that this molecule plays a critical role in capsule biosynthesis and maintaining the cell envelope homeostasis., (© 2019 John Wiley & Sons Ltd.)

Mass spectrometry imaging (MSI) is a technique for mapping the spatial distributions of molecules in sectioned tissue. Histology-preserving tissue preparation methods are central to successful MSI studies. Common fixation methods, used to preserve tissue morphology, can result in artifacts in the resulting MSI experiment including delocalization of analytes, altered adduct profiles, and loss of key analytes due to irreversible cross-linking and diffusion. This is especially troublesome in lung and airway samples, in which histology and morphology is best interpreted from 3D reconstruction, requiring the large and small airways to remain inflated during analysis. Here, we developed an MSI-compatible inflation containing as few exogenous components as possible, forgoing perfusion, fixation, and addition of salt solutions upon inflation that resulted in an ungapped 3D molecular reconstruction through more than 300 microns. We characterized a series of polyunsaturated phospholipids (PUFA-PLs), specifically phosphatidylinositol (-PI) lipids linked to lethal inflammation in bacterial infection and mapped them in serial sections of inflated mouse lung. PUFA-PIs were identified using spatial lipidomics and determined to be determinant markers of major airway features using unsupervised hierarchical clustering. Deep lung architecture was preserved using this inflation approach and the resulting sections are compatible with multiple MSI modalities, automated interpretation software, and serial 3D reconstruction.

Kinase inhibitors comprise a diverse cohort of chemical scaffolds that are active in multiple biological systems. Currently, thousands of eukaryotic kinase inhibitors are commercially available, have well-characterized targets, and often carry pharmaceutically favorable toxicity profiles. Recently, our group disclosed that derivatives of the natural product meridianin D, a known inhibitor of eukaryotic kinases, modulated behaviors of both Gram-positive and Gram-negative bacteria. Herein, we expand our exploration of kinase inhibitors in Gram-negative bacilli utilizing three commercially available kinase inhibitor libraries and, ultimately, identify two chemical structures that potentiate colistin (polymyxin E) in multiple strains. We report IMD-0354, an inhibitor of IKK-β, as a markedly effective adjuvant in colistin-resistant bacteria and also describe AR-12 (OSU-03012), an inhibitor of pyruvate dehydrogenase kinase-1 (PDK-1), as a potentiator in colistin-sensitive strains. This report comprises the first description of the novel cross-reactivity of these molecules.

Burkholderia comprises species that are significant biothreat agents and common contaminants of pharmaceutical production facilities. Their extreme antibiotic resistance affects all classes of antibiotics, including polycationic polymyxins and aminoglycosides. The major underlying mechanism is the presence of two permeability barriers, the outer membrane with modified lipid A moieties and active drug efflux pumps. The two barriers are thought to be mechanistically independent and act synergistically to reduce the intracellular concentrations of antibiotics. In this study, we analyzed the interplay between active efflux pumps and the permeability barrier of the outer membrane in Burkholderia thailandensis We found that three efflux pumps, AmrAB-OprA, BpeEF-OprC, and BpeAB-OprB, of B. thailandensis are expressed under standard laboratory conditions and provide protection against multiple antibiotics, including polycationic polymyxins. Our results further suggest that the inactivation of AmrAB-OprA or BpeAB-OprB potentiates the antibacterial activities of antibiotics not only by reducing their efflux, but also by increasing their uptake into cells. Mass spectrometry analyses showed that in efflux-deficient B. thailandensis cells, lipid A species modified with 4-amino-4-deoxy-l-aminoarabinose are significantly less abundant than in the parent strain. Taken together, our results suggest that changes in the outer membrane permeability due to alterations in lipid A structure could be contributing factors in antibiotic hypersusceptibilities of B. thailandensis cells lacking AmrAB-OprA and BpeAB-OprB efflux pumps., (Copyright © 2019 American Society for Microbiology.)

By circumventing the need for a pure colony, MALDI-TOF mass spectrometry of bacterial membrane glycolipids (lipid A) has the potential to identify microbes more rapidly than protein-based methods. However, currently available bioinformatics algorithms (e.g., dot products) do not work well with glycolipid mass spectra such as those produced by lipid A, the membrane anchor of lipopolysaccharide. To address this issue, we propose a spectral library approach coupled with a machine learning technique to more accurately identify microbes. Here, we demonstrate the performance of the model-based spectral library approach for microbial identification using approximately a thousand mass spectra collected from multi-drug-resistant bacteria. At false discovery rates < 1%, our approach identified many more bacterial species than the existing approaches such as the Bruker Biotyper and characterized over 97% of their phenotypes accurately. As the diversity in our glycolipid mass spectral library increases, we anticipate that it will provide valuable information to more rapidly treat infected patients.

In 2016, the World Health Organization deemed antibiotic resistance one of the biggest threats to global health, food security, and development. The need for new methods to combat infections caused by antibiotic resistant pathogens will require a variety of approaches to identifying effective new therapeutic strategies. One approach is the identification of small molecule adjuvants that potentiate the activity of antibiotics of demonstrated utility, whose efficacy is abated by resistance, both acquired and intrinsic. To this end, we have identified compounds that enhance the efficacy of antibiotics normally ineffective against Gram-negative pathogens because of the outer membrane permeability barrier. We identified two adjuvant compounds that dramatically enhance sensitivity of Acinetobacter baumannii to macrolide and glycopeptide antibiotics, with reductions in minimum inhibitory concentrations as high as 256-fold, and we observed activity across a variety of clinical isolates. Mode of action studies indicate that these adjuvants likely work by modulating lipopolysaccharide synthesis or assembly. The adjuvants were active in vivo in a Galleria mellonella infection model, indicating potential for use in mammalian infections.

The last three decades have seen a dwindling number of novel antibiotic classes approved for clinical use and a concurrent increase in levels of antibiotic resistance, necessitating alternative methods to combat the rise of multi-drug resistant bacteria. A promising strategy employs antibiotic adjuvants, non-toxic molecules that disarm antibiotic resistance. When co-dosed with antibiotics, these compounds restore antibiotic efficacy in drug-resistant strains. Herein we identify derivatives of tryptamine, a ubiquitous biochemical scaffold containing an indole ring system, capable of disarming colistin resistance in the Gram-negative bacterial pathogens Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli while having no inherent bacterial toxicity. Resistance was overcome in strains carrying endogenous chromosomally-encoded colistin resistance machinery, as well as resistance conferred by the mobile colistin resistance-1 (mcr-1) plasmid-borne gene. These compounds restore a colistin minimum inhibitory concentration (MIC) below the Clinical & Laboratory Sciences Institute (CLSI) breakpoint in all resistant strains., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In plants, cell-surface immune receptors sense molecular non-self-signatures. Lipid A of Gram-negative bacterial lipopolysaccharide is considered such a non-self-signature. The receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) mediates plant immune responses to Pseudomonas and Xanthomonas but not enterobacterial lipid A or lipopolysaccharide preparations. Here, we demonstrate that synthetic and bacterial lipopolysaccharide-copurified medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) metabolites elicit LORE-dependent immunity. The mc-3-OH-FAs are sensed in a chain length- and hydroxylation-specific manner, with free ( R )-3-hydroxydecanoic acid [( R )-3-OH-C10:0] representing the strongest immune elicitor. By contrast, bacterial compounds comprising mc-3-OH-acyl building blocks but devoid of free mc-3-OH-FAs-including lipid A or lipopolysaccharide, rhamnolipids, lipopeptides, and acyl-homoserine-lactones-do not trigger LORE-dependent responses. Hence, plants sense low-complexity bacterial metabolites to trigger immune responses., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Acinetobacter baumannii is a prevalent nosocomial pathogen with a high incidence of multidrug resistance. Treatment of infections due to this organism with colistin, a last-resort antibiotic of the polymyxin class, can result in the emergence of colistin-resistant strains. Colistin resistance primarily occurs via modifications of the terminal phosphate moieties of lipopolysaccharide-derived lipid A, which reduces overall membrane electronegativity. These modifications are readily identified by mass spectrometry (MS). In this study, we prospectively collected Acinetobacter baumannii complex clinical isolates from a hospital system in Pennsylvania over a 3-year period. All isolates were evaluated for colistin resistance using standard MIC testing by both agar dilution and broth microdilution, as well as genospecies identification and lipid A profiling using MS analyses. Overall, an excellent correlation between colistin susceptibility and resistance, determined by MIC testing, and the presence of a lipid A modification, determined by MS, was observed with a sensitivity of 92.9% and a specificity of 94.0%. Additionally, glycolipid profiling was able to differentiate A. baumannii complex organisms based on their membrane lipids. With the growth of MS use in clinical laboratories, a reliable MS-based glycolipid phenotyping method that identifies colistin resistance in A. baumannii complex clinical isolates, as well as other Gram-negative organisms, represents an alternative or complementary approach to existing diagnostics., (Copyright © 2019 American Society for Microbiology.)

Pseudomonas aeruginosa is an opportunistic pathogen found ubiquitously in the environment and commonly associated with airway infection in patients with cystic fibrosis. P. aeruginosa strain PAO1 is one of the most commonly used laboratory-adapted research strains and is a standard laboratory-adapted strain in multiple laboratories and strain banks worldwide. Due to potential isolate-to-isolate variability, we investigated the genomic and phenotypic diversity among 10 PAO1 strains (henceforth called sublines) obtained from multiple research laboratories and commercial sources. Genomic analysis predicted a total of 5,682 genes, with 5,434 (95.63%) being identical across all 10 strains. Phenotypic analyses revealed comparable growth phenotypes in rich media and biofilm formation profiles. Limited differences were observed in antibiotic susceptibility profiles and immunostimulatory potential, measured using heat-killed whole-cell preparations in four immortalized cell lines followed by quantification of interleukin-6 (IL-6) and IL-1β secretion. However, variability was observed in the profiles of secreted molecular products, most notably, in rhamnolipid, pyoverdine, pyocyanin, Pseudomonas quinolone signal (PQS), extracellular DNA, exopolysaccharide, and outer membrane vesicle production. Many of the observed phenotypic differences did not correlate with subline-specific genetic changes, suggesting alterations in transcriptional and translational regulation. Taken together, these results suggest that individually maintained sublines of PAO1, even when acquired from the same parent subline, are continuously undergoing microevolution during culture and storage that results in alterations in phenotype, potentially affecting the outcomes of in vitro phenotypic analyses and in vivo pathogenesis studies. IMPORTANCE Laboratory-adapted strains of bacteria are used throughout the world for microbiology research. These prototype strains help keep research data consistent and comparable between laboratories. However, we have observed phenotypic variability when using different strains of Pseudomonas aeruginosa PAO1, one of the major laboratory-adopted research strains. Here, we describe the genomic and phenotypic differences among 10 PAO1 strains acquired from independent sources over 15 years to understand how individual maintenance affects strain characteristics. We observed limited genomic changes but variable phenotypic changes, which may have consequences for cross-comparison of data generated using different PAO1 strains. Our research highlights the importance of limiting practices that may promote the microevolution of model strains and calls for researchers to specify the strain origin to ensure reproducibility., (Copyright © 2019 American Society for Microbiology.)

Infectious diseases have a substantial global health impact. Clinicians need rapid and accurate diagnoses of infections to direct patient treatment and improve antibiotic stewardship. Current technologies employed in routine diagnostics are based on bacterial culture followed by morphological trait differentiation and biochemical testing, which can be time-consuming and labor-intensive. With advances in mass spectrometry (MS) for clinical diagnostics, the U.S. Food and Drug Administration has approved two microbial identification platforms based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis of microbial proteins. We recently reported a novel and complementary approach by comparing MALDI-TOF mass spectra of microbial membrane lipid fingerprints to identify ESKAPE pathogens. However, this lipid-based approach used a sample preparation method that required more than a working day from sample collection to identification. Here, we report a new method that extracts lipids efficiently and rapidly from microbial membranes using an aqueous sodium acetate (SA) buffer that can be used to identify clinically relevant Gram-positive and -negative pathogens and fungal species in less than an hour. The SA method also has the ability to differentiate antibiotic-susceptible and antibiotic-resistant strains, directly identify microbes from biological specimens, and detect multiple pathogens in a mixed sample. These results should have positive implications for the manner in which bacteria and fungi are identified in general hospital settings and intensive care units.

Members of the Rickettsia genus are obligate intracellular, Gram-negative coccobacilli that infect mammalian and arthropod hosts. Several rickettsial species are human pathogens and are transmitted by blood-feeding arthropods. In Gram-negative parasites, the outer membrane (OM) sits at the nexus of the host-pathogen interaction and is rich in lipopolysaccharide (LPS). The lipid A component of LPS anchors the molecule to the bacterial surface and is an endotoxic agonist of Toll-like receptor 4 (TLR4). Despite the apparent importance of lipid A in maintaining OM integrity, as well as its inflammatory potential during infection, this molecule is poorly characterized in Rickettsia pathogens. In this work, we have identified and characterized new members of the recently discovered LpxJ family of lipid A acyltransferases in both Rickettsia typhi and Rickettsia rickettsii , the etiological agents of murine typhus and Rocky Mountain spotted fever, respectively. Our results demonstrate that these enzymes catalyze the addition of a secondary acyl chain (C 14 /C 16 ) to the 3'-linked primary acyl chain of the lipid A moiety in the final steps of the Raetz pathway of lipid A biosynthesis. Since lipid A architecture is fundamental to bacterial OM integrity, we believe that rickettsial LpxJ may be important in maintaining membrane dynamics to facilitate molecular interactions at the host-pathogen interface that are required for adhesion and invasion of mammalian cells. This work contributes to our understanding of rickettsial outer membrane physiology and sets a foundation for further exploration of the envelope and its role in pathogenesis. IMPORTANCE Lipopolysaccharide (LPS) triggers an inflammatory response through the TLR4-MD2 receptor complex and inflammatory caspases, a process mediated by the lipid A moiety of LPS. Species of Rickettsia directly engage both extracellular and intracellular immunosurveillance, yet little is known about rickettsial lipid A. Here, we demonstrate that the alternative lipid A acyltransferase, LpxJ, from Rickettsia typhi and R. rickettsii catalyzes the addition of C 16 fatty acid chains into the lipid A 3'-linked primary acyl chain, accounting for major structural differences relative to the highly inflammatory lipid A of Escherichia coli ., (Copyright © 2018 Guillotte et al.)

Infectious diseases propagated by arthropod vectors, such as tularemia, are commonly initiated via dermal infection of the skin. However, due to the technical difficulties in achieving accurate and reproducible dermal deposition, intradermal models are less commonly used. To overcome these limitations, we used microneedle arrays (MNAs), which are micron-scale polymeric structures, to temporarily disrupt the barrier function of the skin and deliver a bacterial inoculum directly to the dermis of an animal. MNAs increase reliability by eliminating leakage of the inoculum or blood from the injection site, thereby providing a biologically relevant model for arthropod-initiated disease. Here, we validate the use of MNAs as a means to induce intradermal infection using a murine model of tularemia initiated by Francisella novicida We demonstrate targeted delivery of the MNA bolus to the dermal layer of the skin, which subsequently led to innate immune cell infiltration. Additionally, F. novicida -coated MNAs were used to achieve lethality in a dose-dependent manner in C57BL/6 mice. The immune profile of infected mice mirrored that of established F. novicida infection models, consisting of markedly increased serum levels of interleukin-6 and keratinocyte chemoattractant, splenic T-cell depletion, and an increase in splenic granulocytes, together confirming that MNAs can be used to reproducibly induce tularemia-like pathogenesis in mice. When MNAs were used to immunize mice using an attenuated F. novicida mutant ( F. novicida Δ lpxD1 ), all immunized mice survived a lethal subcutaneous challenge. Thus, MNAs can be used to effectively deliver viable bacteria in vivo and provide a novel avenue to study intradermally induced microbial diseases in animal models., (Copyright © 2018 American Society for Microbiology.)

During environmental adaptation bacteria use small regulatory RNAs (sRNAs) to repress or activate expression of a large fraction of their proteome. We extended the use of the in vivo RNA proximity ligation method toward probing global sRNA interactions with their targets in Pseudomonas aeruginosa and verified the method with a known regulon controlled by the PrrF1 sRNA. We also identified two sRNAs (Sr0161 and ErsA) that interact with the mRNA encoding the major porin OprD responsible for the uptake of carbapenem antibiotics. These two sRNAs base pair with the 5' UTR of oprD leading to increase in resistance of the bacteria to meropenem. Additional proximity ligation experiments and enrichment for Sr0161 targets identified the mRNA for the regulator of type III secretion system. Interaction between the exsA mRNA and Sr0161 leads to a block in the synthesis of a component of the T3SS apparatus and an effector. Another sRNA, Sr006, positively regulates, without Hfq, the expression of PagL, an enzyme responsible for deacylation of lipid A, reducing its pro-inflammatory property and resulting in polymyxin resistance. Therefore, an analysis of global sRNA-mRNA interactions can lead to discoveries of novel pathways controlling gene expression that are likely integrated into larger regulatory networks., (© 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

Bacterial resistance to polymyxin antibiotics has taken on a new and more menacing form. Common are genomically-encoded resistance mechanisms to polymyxins, specifically colistin (polymyxin E), however, the plasmid-borne mobile colistin resistance-1 (mcr-1) gene has recently been identified and poses a new threat to global public health. Within six months of initial identification in Chinese swine in November 2015, the first human clinical isolation in the US was reported (Apr. 2016). Herein we report successful reversion of mcr-1-driven colistin resistance in Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli with adjuvants we previously reported as modulators of chromosomally-encoded colistin resistance. Further screening of our in-house library of nitrogen-dense heterocycles has identified additional chemical scaffolds that actively attenuate colistin resistance. Ultimately, we present a diverse cohort of adjuvants that both sensitize colistin-resistant and colistin-susceptible bacteria to this antibiotic, thus providing a potential avenue to both reduce colistin dosage and toxicity, and overcome colistin resistance., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The innate immune system serves as a first line of defense against microbial pathogens. The host innate immune response can be triggered by recognition of conserved non-self-microbial signature molecules by specific host receptor proteins called Toll-like receptors. For bacteria, many of these molecular triggers reside on or are embedded in the bacterial membrane, the interface exposed to the host environment. Lipids are the most abundant component of membranes, and bacteria possess a unique set of lipids that can initiate or modify the host innate immune response. Bacterial lipoproteins, peptidoglycan, and outer membrane molecules lipoteichoic acid and lipopolysaccharide are key modulators of the host immune system. This review article will highlight some of the research emerging at the crossroads of bacterial membranes and innate immunity., Competing Interests: Competing interests: The authors declare that they have no competing interests.No competing interests were disclosed.No competing interests were disclosed.

mcr-1 was initially reported as the first plasmid-mediated colistin resistance gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae in China and has subsequently been identified worldwide in various species of the family Enterobacteriaceae mcr-1 encodes a phosphoethanolamine transferase, and its expression has been shown to generate phosphoethanolamine-modified bis-phosphorylated hexa-acylated lipid A in E. coli Here, we investigated the effects of mcr-1 on colistin susceptibility and on lipopolysaccharide structures in laboratory and clinical strains of the Gram-negative ESKAPE ( Enterococcus faecium , Staphylococcus aureus , K. pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter species) pathogens, which are often treated clinically by colistin. The effects of mcr-1 on colistin resistance were determined using MIC assays of laboratory and clinical strains of E. coli , K. pneumoniae , A. baumannii , and P. aeruginosa Lipid A structural changes resulting from MCR-1 were analyzed by mass spectrometry. The introduction of mcr-1 led to colistin resistance in E. coli , K. pneumoniae , and A. baumannii but only moderately reduced susceptibility in P. aeruginosa Phosphoethanolamine modification of lipid A was observed consistently for all four species. These findings highlight the risk of colistin resistance as a consequence of mcr-1 expression among ESKAPE pathogens, especially in K. pneumoniae and A. baumannii Furthermore, the observation that lipid A structures were modified despite only modest increases in colistin MICs in some instances suggests more sophisticated surveillance methods may need to be developed to track the dissemination of mcr-1 or plasmid-mediated phosphoethanolamine transferases in general., (Copyright © 2017 American Society for Microbiology.)

Rationale: Surface acoustic wave nebulization (SAWN) is an easy to use sample transfer method for rapid mass spectrometric analysis. A new standing wave (SW) SAWN chip, with higher ionization efficiency than our previously reported design, is used for rapid analysis of lipids., Methods: The crude, yet fast, Caroff protocol was used for lipid A extraction from Francisella novicida. SW-SAWN with a Waters Synapt G2S quadrupole time-of-flight (QTOF) mass spectrometer was used to generate lipid A ions. Quadrupole collision-induced dissociation (Q-CID) of lipid A at varying CID energies was used to approximate the ion trap MS n data required for our hierarchical tandem mass spectrometry (HiTMS) algorithm. Structural hypotheses can be obtained directly from the HiTMS algorithm to identify species-specific lipid A molecules., Results: SW-SAWN successfully generated ions from lipid A extracted from Francisella novicida using the faster Caroff method. In addition, varying collision energies were used to generate tandem mass spectra similar to MS 3 and MS 4 spectra from an ion trap. The Q-CID spectra are compatible with our HiTMS algorithm and offer an improvement over lipid A tandem mass spectra acquired in an ion trap., Conclusions: Combining SW-SAWN and Q-CID enabled more structural assignments than previously reported in half the time. The ease of generating spectra by SAWN tandem MS in combination with HiTMS interpretation offers high-throughput lipid A structural analysis and thereby rapid detection of pathogens based on lipid fingerprinting. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Capnocytophaga canimorsus, a commensal bacterium of dog's mouth flora causing severe infections in humans after dog bites or scratches, has a lipopolysaccharide (LPS) (endotoxin) with low-inflammatory lipid A. In particular, it contains a phosphoethanolamine (P-Etn) instead of a free phosphate group at the C-1 position of the lipid A backbone, usually present in highly toxic enterobacterial Gram-negative lipid A. Here we show that the C. canimorsus genome comprises a single operon encoding a lipid A 1-phosphatase (LpxE) and a lipid A 1 P-Etn transferase (EptA). This suggests that lipid A is modified during biosynthesis after completing acylation of the backbone by removal of the 1-phosphate and subsequent addition of an P-Etn group. As endotoxicity of lipid A is known to depend largely on the degree of unsubstituted or unmodified phosphate residues, deletion of lpxE or eptA led to mutants lacking the P-Etn group, with consequently increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP). Consistent with the proposed sequential biosynthetic mechanism, the endotoxicity and CAMP resistance of a double deletion mutant of lpxE-eptA was similar to that of a single lpxE mutant. Finally, the proposed enzymatic activities of LpxE and EptA based on sequence similarity could be successfully validated by mass spectrometry (MS)-based analysis of lipid A isolated from the corresponding deletion mutant strains., (Copyright © 2016 Renzi et al.)

A microsomal triglyceride transfer protein (MTP) inhibitor, CP-346086, was identified that inhibited both human and rodent MTP activity [concentration giving half-maximal inhibition (IC50) 2.0 nM]. In Hep-G2 cells, CP-346086 inhibited apolipoprotein B (apoB) and triglyceride secretion (IC50 2.6 nM) without affecting apoA-I secretion or lipid synthesis. When administered orally to rats or mice, CP-346086 lowered plasma triglycerides [dose giving 30% triglyceride lowering (ED30) 1.3 mg/kg] 2 h after a single dose. Coadministration with Tyloxapol demonstrated that triglyceride lowering was due to inhibition of hepatic and intestinal triglyceride secretion. A 2 week treatment with CP-346086 lowered total, VLDL, and LDL cholesterol and triglycerides dose dependently with 23%, 33%, 75%, and 62% reductions at 10 mg/kg/day. In these animals, MTP inhibition resulted in increased liver and intestinal triglycerides when CP-346086 was administered with food. When dosed away from meals, however, only hepatic triglycerides were increased. When administered as a single oral dose to healthy human volunteers, CP-346086 reduced plasma triglycerides and VLDL cholesterol dose dependently with ED50s of 10 mg and 3 mg, and maximal inhibition (100 mg) of 66% and 87% when measured 4 h after treatment. After a 2 week treatment (30 mg/day), CP-346086 reduced total and LDL cholesterol and triglycerides by 47%, 72%, and 75%, relative to either individual baselines or placebo, with little change in HDL cholesterol. Together, these data support further evaluation of CP-346086 in hyperlipidemia.

The hypocholesterolemic activities of pamaqueside and tiqueside, two structurally similar saponins, were evaluated in cholesterol-fed rabbits. The pharmacological profiles of the saponins were virtually identical: both dose-dependently decreased the intestinal absorption of labeled cholesterol 25-75%, increased fecal neutral sterol excretion up to 2.5-fold, and decreased hepatic cholesterol content 10-55%. High doses of pamaqueside (>5 mg/kg) or tiqueside (>125 mg/kg) completely prevented hypercholesterolemia. Decreases in plasma and hepatic cholesterol levels were strongly correlated with increased neutral sterol excretion. Ratios of neutral sterol excreted to pamaqueside administered were greater than 1:1 at all doses, in opposition to the formation of a stoichiometric complex previously suggested for tiqueside and other saponins. Ratios in tiqueside-treated rabbits were less than unity, a reflection of its lower potency. Pamaqueside-treated rabbits exhibited a more rapid decline in plasma cholesterol concentrations than control animals fed a cholesterol-free diet, indicating that the compound also inhibited the absorption of biliary cholesterol. Intravenous administration of pamaqueside had no effect on plasma cholesterol levels despite plasma levels twice those observed in rabbits given pamaqueside orally. These data indicate that pamaqueside and tiqueside induce hypocholesterolemia by blocking lumenal cholesterol absorption via a mechanism that apparently differs from the stoichiometric complexation of cholesterol hypothesized for other saponins.

We have explored the use of steroidal glycosides as cholesterol absorption inhibitors which act through an unknown mechanism. The lead for this program was tigogenin cellobioside (1, tiqueside) which is a weak inhibitor (ED50 = 60 mg/kg) as measured in an acute hamster cholesterol absorption assay. Modification of the steroid portion of the molecule led to the discovery of 11-ketotigogenin cellobioside (5, pamaqueside) which has an ED50 of 2 mg/kg. Replacement of the cellobiose with other sugars failed to provide more potent analogs. However, large improvements in potency were realized through modification of the hydroxyl groups on the cellobiose. This strategy ultimately led to the 4", 6"-bis[(2-fluorophenyl)carbamoyl]-beta-D-cellobiosyl derivative of 11-ketotigogenin (51) with an ED50 of 0.025 mg/kg in the hamster assay, as well as the corresponding hecogenin analog 64 (ED50 = 0.07 mg/kg).

Bile acids are efficiently recovered from the intestinal lumen by a Na(+)-dependent transport process that is localized in the ileal enterocyte brush-border membrane. To establish a cell culture model for this process, we examined the Na+ dependence of cholyltaurine (C-tau; taurocholate) transport across monolayers of differentiated Caco-2 cells grown on permeable filter inserts. Transport of [3H]C-tau was Na+ dependent (> 20-fold stimulation), saturable, and time linear for at least 60 min. The apparent Michaelis constant of [3H]C-tau transport was approximately 65 microM, and the maximal transport rate was approximately 800 pmol.min-1.mg protein-1. Transport of [3H]C-tau in the apical-to-basolateral direction was 17-fold greater than transport in the reverse direction. Lowered incubation temperature, various metabolic inhibitors, and various unlabeled bile acids inhibited [3H]C-tau transport. Caco-2 cells thus transport bile acids in a manner similar to that described for ileal brush-border membrane vesicles and isolated ileal enterocytes and are therefore an appropriate model for studying the molecular basis of ileal bile acid transport.

Methyl (3R*,5S*)-(E)-3,5-dihydroxy-9,9-diphenyl-6,8-nonadienoate, CP-83101, was identified as a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, inhibiting enzyme activity in vitro with an IC50 of 8.5 +/- 0.7 microM and a Ki with respect to HMG-CoA of 2.6 microM. CP-83101 also inhibited rat hepatic sterol biosynthesis by 39 +/- 7% at a dose of 100 mg/kg. [3H]CP-83101, administered orally to rats, exhibited peak plasma levels at approximately 1 hr that declined thereafter with an apparent half-time of 2-3 hr. Peak tissue levels also occurred 1 hr following oral administration of [3H]CP-83101. The decline in radioactivity in the liver, however, was considerably slower than that noted in blood, whereas the half-life in non-hepatic tissues was approximately 1 hr. Liver/blood ratios of 14, and liver/lens ratios of greater than 3000, following oral administration of [3H]CP-83101, were similar to those previously reported for other HMG-CoA reductase inhibitors, suggesting a high degree of tissue selectivity. In addition, liver/adrenal and liver/ovary ratios were approximately 1000 at all time points examined between 30 min and 24 hr following oral [3H]CP-83101 administration, indicating a high specificity for hepatic versus other steroidogenic tissues. Evaluation of intravenous versus oral administration of the water-soluble, free acid, sodium salt of [3H]CP-83101 in bile duct canulated rats indicated that approximately 20% of orally administered CP-83101 is absorbed from the gastrointestinal tract, and that absorbed CP-83101 is cleared rapidly from the plasma via the liver and from the liver via the bile. In addition, several lines of evidence suggest that CP-83101 may undergo enterohepatic recirculation. Agents of this synthetic series may thus possess advantages over other HMG-CoA reductase inhibitors with respect to tissue kinetics and specificity.

The rat PC12 pheochromocytoma cell line exhibits biological responses to both nerve growth factor (NGF) and epidermal growth factor (EGF). The existence of receptors and biological responses on a common cell for these two well-characterized polypeptide growth factors makes this an attractive system for comparison of ligand binding and processing. Both NGF and EGF are bound to PC12 cells in a competable form at 4 degrees C. At 37 degrees C both ligands are "sequestered," but at different rates and to different extents. While sequestration happens rapidly and nearly quantitatively for bound EGF, the dissociation reaction appears to compete favorably with NGF sequestration. Both EGF and NGF are degraded by PC12 cells. Sequestered EGF, however, is degraded to a greater extent than sequestered NGF.