Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase (PlcH).

Hemolytic phospholipase C, PlcH, is an important virulence factor for Pseudomonas aeruginosa. PlcH preferentially hydrolyzes sphingomyelin and phosphatidylcholine, and this hydrolysis activity drives tissue damage and inflammation and interferes with the oxidative burst of immune cells. Among other contributors, transcription of plcH was previously shown to be induced by phosphate starvation via PhoB and the choline metabolite, glycine betaine, via GbdR. Here, we show that sphingosine can induce plcH transcription and result in secreted PlcH enzyme activity. This induction is dependent on the sphingosine-sensing transcriptional regulator SphR. The SphR induction of plcH occurs from the promoter for the gene upstream of plcH that encodes the neutral ceramidase, CerN, and transcriptional readthrough of the cerN transcription terminator. Evidence for these conclusions came from mutation of the SphR binding site in the cerN promoter, mutation of the cerN terminator, enhancement of cerN termination by adding the rrnB terminator, and reverse transcriptase PCR (RT-PCR) showing that the intergenic region between cerN and plcH is made as RNA during sphingosine, but not choline, induction. We also observed that, like glycine betaine induction, sphingosine induction of plcH is under catabolite repression control, which likely explains why such induction was not seen in other studies using sphingosine in rich media. The addition of sphingosine as a novel inducer for PlcH points to the regulation of plcH transcription as a site for the integration of multiple host-derived signals. IMPORTANCE: PlcH is a secreted phospholipase C/sphingomyelinase that is important for the virulence of Pseudomonas aeruginosa. Here, we show that sphingosine, which presents itself or as a product of P. aeruginosa sphingomyelinase and ceramidase activity, leads to the induction of plcH transcription. This transcriptional induction occurs from the promoter of the upstream ceramidase gene generating a conditional operon. The transcript on which plcH resides, therefore, is different depending on which host molecule or condition leads to induction, and this may have implications for PlcH post-transcriptional regulation. This work also adds to our understanding of P. aeruginosa with host-derived sphingolipids.

Network analysis of patterns and relevance of enteric pathogen co-infections among infants in a diarrhea-endemic setting.

Despite significant progress in recent decades toward ameliorating the excess burden of diarrheal disease globally, childhood diarrhea remains a leading cause of morbidity and mortality in low-and-middle-income countries (LMICs). Recent large-scale studies of diarrhea etiology in these populations have revealed widespread co-infection with multiple enteric pathogens, in both acute and asymptomatic stool specimens. We applied methods from network science and ecology to better understand the underlying structure of enteric co-infection among infants in two large longitudinal birth cohorts in Bangladesh. We used a configuration model to establish distributions of expected random co-occurrence, based on individual pathogen prevalence alone, for every pathogen pair among 30 enteropathogens detected by qRT-PCR in both diarrheal and asymptomatic stool specimens. We found two pairs, Enterotoxigenic E. coli (ETEC) with Enteropathogenic E. coli (EPEC), and ETEC with Campylobacter spp., co-infected significantly more than expected at random (both pairs co-occurring almost 4 standard deviations above what one could expect due to chance alone). Furthermore, we found a general pattern that bacteria-bacteria pairs appear together more frequently than expected at random, while virus-bacteria pairs tend to appear less frequently than expected based on model predictions. Finally, infants co-infected with leading bacteria-bacteria pairs had more days of diarrhea in the first year of life compared to infants without co-infection (p-value <0.0001). Our methods and results help us understand the structure of enteric co-infection which can guide further work to identify and eliminate common sources of infection or determine biologic mechanisms that promote co-infection.

Creatine utilization as a sole nitrogen source in Pseudomonas putida KT2440 is transcriptionally regulated by CahR.

Glutamine amidotransferase-1 domain-containing AraC-family transcriptional regulators (GATRs) are present in the genomes of many bacteria, including all Pseudomonas species. The involvement of several characterized GATRs in amine-containing compound metabolism has been determined, but the full scope of GATR ligands and regulatory networks are still unknown. Here, we characterize Pseudomonas putida's detection of the animal-derived amine compound creatine, a compound particularly enriched in muscle and ciliated cells by a creatine-specific GATR, PP_3665, here named CahR (Creatine amidohydrolase Regulator). cahR is necessary for transcription of the gene encoding creatinase (PP_3667/creA) in the presence of creatine and is critical for P. putida's ability to utilize creatine as a sole source of nitrogen. The CahR/creatine regulon is small, and an electrophoretic mobility shift assay demonstrates strong and specific CahR binding only at the creA promoter, supporting the conclusion that much of the regulon is dependent on downstream metabolites. Phylogenetic analysis of creA orthologues associated with cahR orthologues highlights a strain distribution and organization supporting probable horizontal gene transfer, particularly evident within the genus Acinetobacter. This study identifies and characterizes the GATR that transcriptionally controls P. putida's metabolism of creatine, broadening the scope of known GATR ligands and suggesting GATR diversification during evolution of metabolism for aliphatic nitrogen compounds.

Differential effects of the cystic fibrosis lung inflammatory environment on mesenchymal stromal cells.

Growing evidence demonstrates that human mesenchymal stromal cells (MSCs) modify their in vivo anti-inflammatory actions depending on the specific inflammatory environment encountered. Understanding this better is crucial to refine MSC-based cell therapies for lung and other diseases. Using acute exacerbations of cystic fibrosis (CF) lung disease as a model, the effects of ex vivo MSC exposure to clinical bronchoalveolar lavage fluid (BALF) samples, as a surrogate for the in vivo clinical lung environment, on MSC viability, gene expression, secreted cytokines, and mitochondrial function were compared with effects of BALF collected from healthy volunteers. CF BALF samples that cultured positive for Aspergillus sp. (Asp) induced rapid MSC death, usually within several hours of exposure. Further analyses suggested the fungal toxin gliotoxin as a potential mediator contributing to CF BALF-induced MSC death. RNA sequencing analyses of MSCs exposed to either Asp+ or Asp- CF BALF samples identified a number of differentially expressed transcripts, including those involved in interferon signaling, antimicrobial gene expression, and cell death. Toxicity did not correlate with bacterial lung infections. These results suggest that the potential use of MSC-based cell therapies for CF or other lung diseases may not be warranted in the presence of Aspergillus.

Characterizing species interactions that contribute to biofilm formation in a multispecies model of a potable water bacterial community.

Microbial biofilms are ubiquitous in drinking water systems, yet our understanding of drinking water biofilms lags behind our understanding of those in other environments. Here, a six-member model bacterial community was used to identify the interactions and individual contributions of each species to community biofilm formation. These bacteria were isolated from the International Space Station potable water system and include Cupriavidus metallidurans, Chryseobacterium gleum, Ralstonia insidiosa, Ralstonia pickettii, Methylorubrum (Methylobacterium) populi and Sphingomonas paucimobilis, but all six species are common members of terrestrial potable water systems. Using reconstituted assemblages, from pairs to all 6 members, community biofilm formation was observed to be robust to the absence of any single species and only removal of the C. gleum/S. paucimobilis pair, out of all 15 possible 2-species subtractions, led to loss of community biofilm formation. In conjunction with these findings, dual-species biofilm formation assays supported the view that the contribution of C. gleum to community biofilm formation was dependent on synergistic biofilm formation with either R. insidiosa or C. metallidurans. These data support a model of multiple, partially redundant species interactions to generate robustness in biofilm formation. A bacteriophage and multiple predatory bacteria were used to test the resilience of the community to the removal of individual members in situ, but the combination of precise and substantial depletion of a single target species was not achievable. We propose that this assemblage can be used as a tractable model to understand the molecular bases of the interactions described here and to decipher other functions of drinking water biofilms.

Bacterial Lipoproteins Constitute the TLR2-Stimulating Activity of Serum Amyloid A.

Studies comparing endogenous and recombinant serum amyloid A (SAA) have generated conflicting data on the proinflammatory function of these proteins. In exploring this discrepancy, we found that in contrast to commercially sourced recombinant human SAA1 (hSAA1) proteins produced in Escherichia coli, hSAA1 produced from eukaryotic cells did not promote proinflammatory cytokine production from human or mouse cells, induce Th17 differentiation, or stimulate TLR2. Proteomic analysis of E. coli-derived hSAA1 revealed the presence of numerous bacterial proteins, with several being reported or probable lipoproteins. Treatment of hSAA1 with lipoprotein lipase or addition of a lipopeptide to eukaryotic cell-derived hSAA1 inhibited or induced the production of TNF-α from macrophages, respectively. Our results suggest that a function of SAA is in the binding of TLR2-stimulating bacterial proteins, including lipoproteins, and demand that future studies of SAA employ a recombinant protein derived from eukaryotic cells.

Hybrid Model of Bacterial Biofilm Growth.

Bacterial biofilms play a critical role in environmental processes, water treatment, human health, and food processing. They exhibit highly complex dynamics due to the interactions between the bacteria and the extracellular polymeric substance (EPS), water, and nutrients and minerals that make up the biofilm. We present a hybrid computational model in which the dynamics of discrete bacterial cells are simulated within a multiphase continuum, consisting of EPS and water as separate interacting phases, through which nutrients and minerals diffuse. Bacterial cells in our model consume water and nutrients in order to grow, divide, and produce EPS. Consequently, EPS flows outward from the bacterial colony, while water flows inward. The model predicts bacterial colony formation as a treelike structure. The distribution of bacterial growth and EPS production is found to be sensitive to the pore spacing between bacteria and the consumption of nutrients within the bacterial colony. Forces that are sometimes neglected in biofilm simulations, such as lubrication force between nearby bacterial cells and osmotic (swelling) pressure force resulting from gradients in EPS concentration, are observed to have an important effect on biofilm growth via their influence on bacteria pore spacing and associated water/nutrient percolation into the bacterial colony.

Stenotrophomonas maltophilia Differential Gene Expression in Synthetic Cystic Fibrosis Sputum Reveals Shared and Cystic Fibrosis Strain-Specific Responses to the Sputum Environment.

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can infect the lungs of people with cystic fibrosis (CF). The highly viscous mucus in the CF lung, expectorated as sputum, serves as the primary nutrient source for microbes colonizing this site and induces virulence-associated phenotypes and gene expression in several CF pathogens. Here, we characterized the transcriptional responses of three S. maltophilia strains during exposure to synthetic CF sputum medium (SCFM2) to gain insight into how this organism interacts with the host in the CF lung. These efforts led to the identification of 881 transcripts differentially expressed by all three strains, many of which reflect the metabolic pathways used by S. maltophilia in sputum, as well as altered stress responses. The latter correlated with increased resistance to peroxide exposure after pregrowth in SCFM2 for two of the strains. We also compared the SCFM2 transcriptomes of two S. maltophilia CF isolates to that of the acute infection strain, S. maltophilia K279a, allowing us to identify CF isolate-specific signatures in differential gene expression. The expression of genes from the accessory genomes was also differentially altered in response to SCFM2. Finally, a number of biofilm-associated genes were differentially induced in SCFM2, particularly in K279a, which corresponded to increased aggregation and biofilm formation in this strain relative to both CF strains. Collectively, this work details the response of S. maltophilia to an environment that mimics important aspects of the CF lung, identifying potential survival strategies and metabolic pathways used by S. maltophilia during infections.IMPORTANCEStenotrophomonas maltophilia is an important infecting bacterium in the airways of people with cystic fibrosis (CF). However, compared to the other CF pathogens, S. maltophilia has been relatively understudied. The significance of our research is to provide insight into the global transcriptomic changes of S. maltophilia in response to a medium that was designed to mimic important aspects of the CF lung. This study elucidates the overall metabolic changes that occur when S. maltophilia encounters the CF lung and generates a road map of candidate genes to test using in vitro and in vivo models of CF.

Lung inflammatory environments differentially alter mesenchymal stromal cell behavior.

Mesenchymal stromal (stem) cells (MSCs) are increasingly demonstrated to ameliorate experimentally induced lung injuries through disease-specific anti-inflammatory actions, thus suggesting that different in vivo inflammatory environments can influence MSC actions. To determine the effects of different representative inflammatory lung conditions, human bone marrow-derived MSCs (hMSCs) were exposed to in vitro culture conditions from bronchoalveolar lavage fluid (BALF) samples obtained from patients with either the acute respiratory distress syndrome (ARDS) or with other lung diseases including acute respiratory exacerbations of cystic fibrosis (CF) (non-ARDS). hMSCs were subsequently assessed for time- and BALF concentration-dependent effects on mRNA expression of selected pro- and anti-inflammatory mediators, and for overall patterns of gene and mRNA expression. Both common and disease-specific patterns were observed in gene expression of different hMSC mediators, notably interleukin (IL)-6. Conditioned media obtained from non-ARDS BALF-exposed hMSCs was more effective in promoting an anti-inflammatory phenotype in monocytes than was conditioned media from ARDS BALF-exposed hMSCs. Neutralizing IL-6 in the conditioned media promoted generation of anti-inflammatory monocyte phenotype. This proof of concept study suggest that different lung inflammatory environments potentially can alter hMSC behaviors. Further identification of these interactions and the driving mechanisms may influence clinical use of MSCs for treating lung diseases.

Breath metabolome of mice infected with Pseudomonas aeruginosa.

INTRODUCTION: The measurement of specific volatile organic compounds in breath has been proposed as a potential diagnostic for a variety of diseases. The most well-studied bacterial lung infection in the breath field is that caused by Pseudomonas aeruginosa. OBJECTIVES: To determine a discriminatory core of molecules in the "breath-print" of mice during a lung infection with four strains of P. aeruginosa (PAO1, PA14, PAK, PA7). Furthermore, we attempted to extrapolate a strain-specific "breath-print" signature to investigate the possibility of recapitulating the genetic phylogenetic groups (Stewart et al. Pathog Dis 71(1), 20-25, 2014. https://doi.org/10.1111/2049-632X.12107 ). METHODS: Breath was collected into a Tedlar bag and shortly after drawn into a thermal desorption tube. The latter was then analyzed into a comprehensive multidimensional gas chromatography coupled with a time-of-flight mass spectrometer. Random forest algorithm was used for selecting the most discriminatory features and creating a prediction model. RESULTS: Three hundred and one molecules were significantly different between animals infected with P. aeruginosa, and those given a sham infection (PBS) or inoculated with UV-killed P. aeruginosa. Of those, nine metabolites could be used to discriminate between the three groups with an accuracy of 81%. Hierarchical clustering showed that the signature from breath was due to a specific response to live bacteria instead of a generic infection response. Furthermore, we identified ten additional volatile metabolites that could differentiate mice infected with different strains of P. aeruginosa. A phylogram generated from the ten metabolites showed that PAO1 and PA7 were the most distinct group, while PAK and PA14 were interspersed between the former two groups. CONCLUSIONS: To the best of our knowledge, this is the first study to report on a 'core' murine breath print, as well as, strain level differences between the compounds in breath. We provide identifications (by running commercially available analytical standards) to five breath compounds that are predictive of P. aeruginosa infection.

Pulmonary Surfactant Promotes Virulence Gene Expression and Biofilm Formation in Klebsiella pneumoniae.

The interactions between Klebsiella pneumoniae and the host environment at the site of infection are largely unknown. Pulmonary surfactant serves as an initial point of contact for inhaled bacteria entering the lung and is thought to contain molecular cues that aid colonization and pathogenesis. To gain insight into this ecological transition, we characterized the transcriptional response of K. pneumoniae MGH 78578 to purified pulmonary surfactant. This work revealed changes within the K. pneumoniae transcriptome that likely contribute to host colonization, adaptation, and virulence in vivo Notable transcripts expressed under these conditions include genes involved in capsule synthesis, lipopolysaccharide modification, antibiotic resistance, biofilm formation, and metabolism. In addition, we tested the contributions of other surfactant-induced transcripts to K. pneumoniae survival using engineered isogenic KPPR1 deletion strains in a murine model of acute pneumonia. In these infection studies, we identified the MdtJI polyamine efflux pump and the ProU glycine betaine ABC transporter to be significant mediators of K. pneumoniae survival within the lung and confirmed previous evidence for the importance of de novo leucine synthesis to bacterial survival during infection. Finally, we determined that pulmonary surfactant promoted type 3 fimbria-mediated biofilm formation in K. pneumoniae and identified two surfactant constituents, phosphatidylcholine and cholesterol, that drive this response. This study provides novel insight into the interactions occurring between K. pneumoniae and the host at an important infection site and demonstrates the utility of purified lung surfactant preparations for dissecting host-lung pathogen interactions in vitro.

Differential requirements for processing and transport of short-chain versus long-chain O-acylcarnitines in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa can metabolize carnitine and O-acylcarnitines, which are abundant in host muscle and other tissues. Acylcarnitines are metabolized to carnitine and a fatty acid. The liberated carnitine and its catabolic product, glycine betaine, can be used as osmoprotectants, to induce the secreted phospholipase C PlcH, and as sole carbon, nitrogen and energy sources. P. aeruginosa is incapable of de novo synthesis of carnitine and acylcarnitines, therefore they must be imported from an exogenous source. In this study, we present the first characterization of bacterial acylcarnitine transport. Short-chain acylcarnitines are imported by the ABC transporter CaiX-CbcWV. Medium- and long-chain acylcarnitines (MCACs and LCACs) are hydrolysed extracytoplasmically and the free carnitine is transported primarily through CaiX-CbcWV. These findings suggest that the periplasmic protein CaiX has a binding pocket that permits short acyl chains on its carnitine ligand and that there are one or more secreted hydrolases that cleave MCACs and LCACs. To identify the secreted hydrolase(s), we used a saturating genetic screen and transcriptomics followed by phenotypic analyses, but neither led to identification of a contributing hydrolase, supporting but not conclusively demonstrating redundancy for this activity.

Transcriptional Responses of Pseudomonas aeruginosa to Potable Water and Freshwater.

Many Pseudomonas aeruginosa infections are derived from residential, recreational, or surface water sources; thus, these environments represent an important preinfection niche. To better understand P. aeruginosa biology in these environments, we quantified transcriptional changes by microarray after exposure to diluted LB, diluted R2B, potable tap water, and freshwater from a eutrophic pond. Quantitative reverse transcription-PCR (qRT-PCR) confirmed the conservation of these responses in other water sources, and competition experiments were used to test the importance of three implicated metabolic pathways. The global transcriptional responses in potable water and freshwater showed strong induction of genes involved in metabolism of the head groups and acyl tails of phospholipids, as well as nucleotide metabolism, with commensurate decreased transcript expression of genes encoding their synthetic pathways. These data suggest that phospholipids and nucleotides are part of the nutritional milieu of these two environments. A unique response in municipal-delivered potable water was to the metals in the piping system, particularly copper. To identify potential nutrient sources used by P. aeruginosa in these environments, we used competition assays between the wild-type and deletion mutant strains in three pathways induced under these conditions. For phospholipid head-group metabolism, ethanolamine utilization (eutB) was important for competition in potable water, while choline oxidation (betBA) was important for competition in freshwater. Nucleotide utilization, particularly pyrimidine metabolism (dht), showed a trend toward importance in freshwater but was not statistically significant. These findings provide new insights into the P. aeruginosa response to potable water and freshwater and led to the identification of potentially important nutrient sources in these environments.IMPORTANCE Much of our knowledge about Pseudomonas aeruginosa comes from the infection niche, and much less is known about its lifestyle in the environment. P. aeruginosa is an adaptable bacterium capable of growing in many environments but is particularly common in potable water systems and freshwater. We used the transcriptional responses of P. aeruginosa to these environments to identify important nutrient sources specific to either of these two environments. Additionally, these environments could provide experimental situations to understand gene function for the large number of transcripts with unknown functions induced under these conditions.

Choline Catabolism in Burkholderia thailandensis Is Regulated by Multiple Glutamine Amidotransferase 1-Containing AraC Family Transcriptional Regulators.

UNLABELLED: Burkholderia thailandensis is a soil-dwelling bacterium that shares many metabolic pathways with the ecologically similar, but evolutionarily distant, Pseudomonas aeruginosa Among the diverse nutrients it can utilize is choline, metabolizable to the osmoprotectant glycine betaine and subsequently catabolized as a source of carbon and nitrogen, similar to P. aeruginosa Orthologs of genes in the choline catabolic pathway in these two bacteria showed distinct differences in gene arrangement as well as an additional orthologous transcriptional regulator in B. thailandensis In this study, we showed that multiple glutamine amidotransferase 1 (GATase 1)-containing AraC family transcription regulators (GATRs) are involved in regulation of the B. thailandensis choline catabolic pathway (gbdR1, gbdR2, and souR). Using genetic analyses and sequencing the transcriptome in the presence and absence of choline, we identified the likely regulons of gbdR1 (BTH_II1869) and gbdR2 (BTH_II0968). We also identified a functional ortholog for P. aeruginosa souR, a GATR that regulates the metabolism of sarcosine to glycine. GbdR1 is absolutely required for expression of the choline catabolic locus, similar to P. aeruginosa GbdR, while GbdR2 is important to increase expression of the catabolic locus. Additionally, the B. thailandensis SouR ortholog (BTH_II0994) is required for catabolism of choline and its metabolites as carbon sources, whereas in P. aeruginosa, SouR function can by bypassed by GbdR. The strategy employed by B. thailandensis represents a distinct regulatory solution to control choline catabolism and thus provides both an evolutionary counterpoint and an experimental system to analyze the acquisition and regulation of this pathway during environmental growth and infection. IMPORTANCE: Many proteobacteria that occupy similar environmental niches have horizontally acquired orthologous genes for metabolism of compounds useful in their shared environment. The arrangement and differential regulation of these components can help us understand both the evolution of these systems and the potential roles these pathways have in the biology of each bacterium. Here, we describe the transcriptome response of Burkholderia thailandensis to the eukaryote-enriched molecule choline, identify the regulatory pathway governing choline catabolism, and compare the pathway to that previously described for Pseudomonas aeruginosa These data support a multitiered regulatory network in B. thailandensis, with conserved orthologs in the select agents Burkholderia pseudomallei and Burkholderia mallei, as well as the opportunistic lung pathogens in the Burkholderia cepacia clade.

Sarcosine Catabolism in Pseudomonas aeruginosa Is Transcriptionally Regulated by SouR.

UNLABELLED: Sarcosine (N-methylglycine) is present in many environments inhabited by pseudomonads and is likely most often encountered as an intermediate in the metabolism of choline, carnitine, creatine, and glyphosate. While the enzymology of sarcosine metabolism has been relatively well studied in bacteria, the regulatory mechanisms governing catabolism have remained largely unknown. We previously determined that the sarcosine-catabolic (sox) operon of Pseudomonas aeruginosa is induced by the AraC family regulator GbdR in response to glycine betaine and dimethylglycine. However, induction of these genes was still observed in response to sarcosine in a gbdR deletion mutant, indicating that an independent sarcosine-responsive transcription factor also acted at this locus. Our goal in this study was to identify and characterize this regulator. Using a transposon-based genetic screen, we identified PA4184, or SouR (sarcosine oxidation and utilization regulator), as the sarcosine-responsive regulator of the sox operon, with tight induction specificity for sarcosine. The souR gene is required for appreciable growth on sarcosine as a carbon and nitrogen source. We also characterized the transcriptome response to sarcosine governed by SouR using microarray analyses and performed electrophoretic mobility shift assays to identify promoters directly regulated by the transcription factor. Finally, we characterized PA3630, or GfnR (glutathione-dependent formaldehyde neutralization regulator), as the regulator of the glutathione-dependent formaldehyde detoxification system in P. aeruginosa that is expressed in response to formaldehyde released during the catabolism of sarcosine. This study expands our understanding of sarcosine metabolic regulation in bacteria through the identification and characterization of the first known sarcosine-responsive transcriptional regulator. IMPORTANCE: The Pseudomonas aeruginosa genome encodes many diverse metabolic pathways, yet the specific transcription regulators controlling their expression remain mostly unknown. Here, we used a genetic screen to identify the sarcosine-specific regulator of the sarcosine oxidase operon, which we have named SouR. SouR is the first bacterial regulator shown to respond to sarcosine, and it is required for growth on sarcosine. Sarcosine is found in its free form and is also an intermediate in the catabolic pathways of glycine betaine, carnitine, creatine, and glyphosate. The similarity of SouR to the regulators of carnitine and glycine betaine catabolism suggests evolutionary diversification within this regulatory family to allow response to structurally similar but physiologically distinct ligands.

A Comparative Study of Lung Host Defense in Murine Obesity Models. Insights into Neutrophil Function.

We have shown that obesity-associated attenuation of murine acute lung injury is driven, in part, by blunted neutrophil chemotaxis, yet differences were noted between the two models of obesity studied. We hypothesized that obesity-associated impairment of multiple neutrophil functions contributes to increased risk for respiratory infection but that such impairments may vary between murine models of obesity. We examined the most commonly used murine obesity models (diet-induced obesity, db/db, CPE(fat/fat), and ob/ob) using a Klebsiella pneumoniae pneumonia model and LPS-induced pneumonitis. Marrow-derived neutrophils from uninjured lean and obese mice were examined for in vitro functional responses. All obesity models showed impaired clearance of K. pneumoniae, but in differing temporal patterns. Failure to contain infection in obese mice was seen in the db/db model at both 24 and 48 hours, yet this defect was only evident at 24 hours in CPE(fat/fat) and ob/ob models, and at 48 hours in diet-induced obesity. LPS-induced airspace neutrophilia was decreased in all models, and associated with blood neutropenia in the ob/ob model but with leukocytosis in the others. Obese mouse neutrophils from all models demonstrated impaired chemotaxis, whereas neutrophil granulocyte colony-stimulating factor-mediated survival, LPS-induced cytokine transcription, and mitogen-activated protein kinase and signal transducer and activator of transcription 3 activation in response to LPS and granulocyte colony-stimulating factor, respectively, were variably impaired across the four models. Obesity-associated impairment of host response to lung infection is characterized by defects in neutrophil recruitment and survival. However, critical differences exist between commonly used mouse models of obesity and may reflect variable penetrance of elements of the metabolic syndrome, as well as other factors.

The Type Three Secretion System 2-Encoded Regulator EtrB Modulates Enterohemorrhagic Escherichia coli Virulence Gene Expression.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a foodborne pathogen that causes bloody diarrhea and hemolytic uremic syndrome throughout the world. A defining feature of EHEC pathogenesis is the formation of attaching and effacing (AE) lesions on colonic epithelial cells. Most of the genes that code for AE lesion formation, including a type three secretion system (T3SS) and effectors, are carried within a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE). In this study, we report that a putative regulator, which is encoded in the cryptic E. coli type three secretion system 2 (ETT2) locus and herein renamed EtrB, plays an important role in EHEC pathogenesis. The etrB gene is expressed as a monocistronic transcript, and EtrB autoregulates expression. We provide evidence that EtrB directly interacts with the ler regulatory region to activate LEE expression and promote AE lesion formation. Additionally, we mapped the EtrB regulatory circuit in EHEC to determine a global role for EtrB. EtrB is regulated by the transcription factor QseA, suggesting that these proteins comprise a regulatory circuit important for EHEC colonization of the gastrointestinal tract.

Detection of host-derived sphingosine by Pseudomonas aeruginosa is important for survival in the murine lung.

Pseudomonas aeruginosa is a common environmental bacterium that is also a significant opportunistic pathogen, particularly of the human lung. We must understand how P. aeruginosa responds to the lung environment in order to identify the regulatory changes that bacteria use to establish and maintain infections. The P. aeruginosa response to pulmonary surfactant was used as a model to identify transcripts likely induced during lung infection. The most highly induced transcript in pulmonary surfactant, PA5325 (sphA), is regulated by an AraC-family transcription factor, PA5324 (SphR). We found that sphA was specifically induced by sphingosine in an SphR-dependent manner, and also via metabolism of sphingomyelin, ceramide, or sphingoshine-1-phosphate to sphingosine. These sphingolipids not only play a structural role in lipid membranes, but some are also intracellular and intercellular signaling molecules important in normal eukaryotic cell functions as well as orchestrating immune responses. The members of the SphR transcriptome were identified by microarray analyses, and DNA binding assays showed specific interaction of these promoters with SphR, which enabled us to determine the consensus SphR binding site. SphR binding to DNA was modified by sphingosine and we used labeled sphingosine to demonstrate direct binding of sphingosine by SphR. Deletion of sphR resulted in reduced bacterial survival during mouse lung infection. In vitro experiments show that deletion of sphR increases sensitivity to the antimicrobial effects of sphingosine which could, in part, explain the in vivo phenotype. This is the first identification of a sphingosine-responsive transcription factor in bacteria. We predict that SphR transcriptional regulation may be important in response to many sites of infection in eukaryotes and the presence of homologous transcription factors in other pathogens suggests that sphingosine detection is not limited to P. aeruginosa.

The role of CD1d-restricted NKT cells in the clearance of Pseudomonas aeruginosa from the lung is dependent on the host genetic background.

Pseudomonas aeruginosa is an important human opportunistic pathogen, accounting for a significant fraction of hospital-acquired lung infections. CD1d-restricted NKT cells comprise an unusual innate-like T cell subset that plays important roles in both bacterial and viral infections. Previous reports have differed in their conclusions regarding the role of NKT cells in clearance of P. aeruginosa from the lung. Since there is significant strain-dependent variation in NKT cell number and function among different inbred strains of mice, we investigated whether the role of NKT cells was dependent on the host genetic background. We found that NKT cells did indeed play a critical role in the clearance of P. aeruginosa from the lungs of BALB/c mice but that they played no discernible role in clearance from the lungs of C57BL/6 mice. We found that the strain-dependent role of NKT cells was associated with significant strain-dependent differences in cytokine production by lung NKT cells and that impaired clearance of P. aeruginosa in BALB/c CD1d(-/-) mice was associated with an increase in neutrophil influx to the lung and increased levels of proinflammatory cytokines and chemokines after infection. Finally, we found that the role of alveolar macrophages was also dependent on the genetic background. These data provide further support for a model in which the unusually high level of variability in NKT cell number and function among different genetic backgrounds may be an important contributor to infectious-disease susceptibility and pathology.

Carnitine in bacterial physiology and metabolism.

Carnitine is a quaternary amine compound found at high concentration in animal tissues, particularly muscle, and is most well studied for its contribution to fatty acid transport into mitochondria. In bacteria, carnitine is an important osmoprotectant, and can also enhance thermotolerance, cryotolerance and barotolerance. Carnitine can be transported into the cell or acquired from metabolic precursors, where it can serve directly as a compatible solute for stress protection or be metabolized through one of a few distinct pathways as a nutrient source. In this review, we summarize what is known about carnitine physiology and metabolism in bacteria. In particular, recent advances in the aerobic and anaerobic metabolic pathways as well as the use of carnitine as an electron acceptor have addressed some long-standing questions in the field.