Global stress response in Pseudomonas aeruginosa upon malonate utilization.

Versatility in carbon source utilization assists Pseudomonas aeruginosa in its adaptation to various niches. Recently, we characterized the role of malonate, an understudied carbon source, in quorum sensing regulation, antibiotic resistance, and virulence factor production in P. aeruginosa . These results indicate that global responses to malonate metabolism remain to be uncovered. We leveraged a publicly available metabolomic dataset on human airway and found malonate to be as abundant as glycerol, a common airway metabolite and carbon source for P. aeruginosa . Here, we explored and compared adaptations of P. aeruginosa UCBPP-PA14 (PA14) in response to malonate or glycerol as a sole carbon source using transcriptomics and phenotypic assays. Malonate utilization activated glyoxylate and methylcitrate cycles and induced several stress responses, including oxidative, anaerobic, and metal stress responses associated with increases in intracellular aluminum and strontium. Some induced genes were required for optimal growth of P. aeruginosa in malonate. To assess the conservation of malonate-associated responses among P. aeruginosa strains, we compared our findings in strain PA14 with other lab strains and cystic fibrosis isolates of P. aeruginosa . Most strains grew on malonate as a sole carbon source as efficiently as or better than glycerol. While not all responses to malonate were conserved among strains, formation of biomineralized biofilm-like aggregates, increased tolerance to kanamycin, and increased susceptibility to norfloxacin were the most frequently observed phenotypes. Our findings reveal global remodeling of P. aeruginosa gene expression during its growth on malonate as a sole carbon source that is accompanied by several important phenotypic changes. These findings add to accumulating literature highlighting the role of different carbon sources in the physiology of P. aeruginosa and its niche adaptation. Importance: Pseudomonas aeruginosa is a notorious pathogen that causes local and systemic infections in immunocompromised individuals. Different carbon sources can uniquely modulate metabolic and virulence pathways in P. aeruginosa , highlighting the importance of the environment that the pathogen occupies. In this work, we used a combination of transcriptomic analysis and phenotypic assays to determine how malonate utilization impacts P. aeruginosa, as recent evidence indicates this carbon source may be relevant to certain niches associated within the human host. We found that malonate utilization can induce global stress responses, alter metabolic circuits, and influence various phenotypes of P. aeruginosa that could influence host colonization. Investigating the metabolism of malonate provides insight into P. aeruginosa adaptations to specific niches where this substrate is abundant, and how it can be leveraged in the development of much-needed antimicrobial agents or identification of new therapeutic targets of this difficult-to-eradicate pathogen.

Glycogen availability and pH variation in a medium simulating vaginal fluid influence the growth of vaginal Lactobacillus species and Gardnerella vaginalis.

BACKGROUND: Glycogen metabolism by Lactobacillus spp. that dominate the healthy vaginal microbiome contributes to a low vaginal pH (3.5-4.5). During bacterial vaginosis (BV), strict and facultative anaerobes including Gardnerella vaginalis become predominant, leading to an increase in the vaginal pH (> 4.5). BV enhances the risk of obstetrical complications, acquisition of sexually transmitted infections, and cervical cancer. Factors critical for the maintenance of the healthy vaginal microbiome or the transition to the BV microbiome are not well defined. Vaginal pH may affect glycogen metabolism by the vaginal microflora, thus influencing the shift in the vaginal microbiome. RESULTS: The medium simulating vaginal fluid (MSVF) supported growth of L. jensenii 62G, L. gasseri 63 AM, and L. crispatus JV-V01, and G. vaginalis JCP8151A at specific initial pH conditions for 30 d. L. jensenii at all three starting pH levels (pH 4.0, 4.5, and 5.0), G. vaginalis at pH 4.5 and 5.0, and L. gasseri at pH 5.0 exhibited the long-term stationary phase when grown in MSVF. L. gasseri at pH 4.5 and L. crispatus at pH 5.0 displayed an extended lag phase over 30 d suggesting inefficient glycogen metabolism. Glycogen was essential for the growth of L. jensenii, L. crispatus, and G. vaginalis; only L. gasseri was able to survive in MSVF without glycogen, and only at pH 5.0, where it used glucose. All four species were able to survive for 15 d in MSVF with half the glycogen content but only at specific starting pH levels - pH 4.5 and 5.0 for L. jensenii, L. gasseri, and G. vaginalis and pH 5.0 for L. crispatus. CONCLUSIONS: These results suggest that variations in the vaginal pH critically influence the colonization of the vaginal tract by lactobacilli and G. vaginalis JCP8151A by affecting their ability to metabolize glycogen. Further, we found that L. jensenii 62G is capable of glycogen metabolism over a broader pH range (4.0-5.0) while L. crispatus JV-V01 glycogen utilization is pH sensitive (only functional at pH 5.0). Finally, our results showed that G. vaginalis JCP8151A can colonize the vaginal tract for an extended period as long as the pH remains at 4.5 or above.

Anti-Pseudomonas aeruginosa Vaccines and Therapies: An Assessment of Clinical Trials.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes high morbidity and mortality in cystic fibrosis (CF) and immunocompromised patients, including patients with ventilator-associated pneumonia (VAP), severely burned patients, and patients with surgical wounds. Due to the intrinsic and extrinsic antibiotic resistance mechanisms, the ability to produce several cell-associated and extracellular virulence factors, and the capacity to adapt to several environmental conditions, eradicating P. aeruginosa within infected patients is difficult. Pseudomonas aeruginosa is one of the six multi-drug-resistant pathogens (ESKAPE) considered by the World Health Organization (WHO) as an entire group for which the development of novel antibiotics is urgently needed. In the United States (US) and within the last several years, P. aeruginosa caused 27% of deaths and approximately USD 767 million annually in health-care costs. Several P. aeruginosa therapies, including new antimicrobial agents, derivatives of existing antibiotics, novel antimicrobial agents such as bacteriophages and their chelators, potential vaccines targeting specific virulence factors, and immunotherapies have been developed. Within the last 2-3 decades, the efficacy of these different treatments was tested in clinical and preclinical trials. Despite these trials, no P. aeruginosa treatment is currently approved or available. In this review, we examined several of these clinicals, specifically those designed to combat P. aeruginosa infections in CF patients, patients with P. aeruginosa VAP, and P. aeruginosa-infected burn patients.

Osteoprotective effect of green tea polyphenols and annatto-extracted tocotrienol in obese mice is associated with enhanced microbiome vitamin K2 biosynthetic pathways.

The role of the gut microbiome in bone health has received significant attention in the past decade. We investigated the effects of green tea polyphenols (GTP) and annatto-extracted tocotrienols (AT) on bone properties and gut microbiome in obese mice. Male mice were assigned to a two (no AT vs. 400 mg/kg diet AT) × two (no GTP vs. 0.5% w/v GTP) factorial design, namely control, G, T, and G+T group respectively, for 14 weeks. The 4th lumbar vertebra (LV-4) and femur were harvested for bone microstructural analysis using µ-CT. Microbiome analysis using 16S rRNA gene sequencing of cecal feces was performed. AT increased bone volume at distal femur. GTP increased serum procollagen type 1 N-terminal propeptide concentration, bone volume at the distal femur and the LV-4, and trabecular number at distal femur; whereas GTP decreased trabecular separation at distal femur. Interactions between GTP and AT were observed in serum C-terminal telopeptide of type I collagen level (control>G=T=G+T) as well as the cortical bone area (control

Hereditary hemochromatosis promotes colitis and colon cancer and causes bacterial dysbiosis in mice.

Hereditary hemochromatosis (HH), an iron-overload disease, is a prevalent genetic disorder. As excess iron causes a multitude of metabolic disturbances, we postulated that iron overload in HH disrupts colonic homeostasis and colon-microbiome interaction and exacerbates the development and progression of colonic inflammation and colon cancer. To test this hypothesis, we examined the progression and severity of colitis and colon cancer in a mouse model of HH (Hfe-/-), and evaluated the potential contributing factors. We found that experimentally induced colitis and colon cancer progressed more robustly in Hfe-/- mice than in wild-type mice. The underlying causes were multifactorial. Hfe-/- colons were leakier with lower proliferation capacity of crypt cells, which impaired wound healing and amplified inflammation-driven tissue injury. The host/microflora axis was also disrupted. Sequencing of fecal 16S RNA revealed profound changes in the colonic microbiome in Hfe-/- mice in favor of the pathogenic bacteria belonging to phyla Proteobacteria and TM7. There was an increased number of bacteria adhered onto the mucosal surface of the colonic epithelium in Hfe-/- mice than in wild-type mice. Furthermore, the expression of innate antimicrobial peptides, the first-line of defense against bacteria, was lower in Hfe-/- mouse colon than in wild-type mouse colon; the release of pro-inflammatory cytokines upon inflammatory stimuli was also greater in Hfe-/- mouse colon than in wild-type mouse colon. These data provide evidence that excess iron accumulation in colonic tissue as happens in HH promotes colitis and colon cancer, accompanied with bacterial dysbiosis and loss of function of the intestinal/colonic barrier.

Organoselenium Polymer Inhibits Biofilm Formation in Polypropylene Contact Lens Case Material.

OBJECTIVES: Contact lens-acquired bacterial infections are a serious problem. Of the reported cases, inadequate cleaning of the lens case was the most common cause of lens contamination. Organoselenium has been shown to inhibit bacterial attachment to different polymer materials. This study evaluates the ability of an organoselenium monomer, incorporated into the polymer of a polypropylene contact lens case coupon, to block the formation of biofilms in a lens case. METHODS: The bacteria tested were Pseudomonas aeruginosa, Staphylococcus aureus, Stenotrophomonas maltophilia, and Serratia marcescens. For this study, the bacteria were allowed to grow overnight, in trypticase soy broth media, in the presence of the selenium-containing polymer or the same polymer without organoselenium. The material was studied by both colony-forming unit determination and by confocal laser scanning microscopy. RESULTS: The results showed that the organoselenium polymer versus the control polymer resulted in the following effect on biofilm formation: (1) a reduction in P. aeruginosa of 7.3 logs (100%); (2) a reduction in S. aureus of 7.3 logs (100%); (3) a reduction in S. maltophilia of 7.5 logs (100%); and (4) a reduction in S. marcescens reduction of 3.3 logs (99.9%). To test the stability of the organoselenium polypropylene contact lens coupon, the coupon was soaked in PBS for eight weeks at room temperature. It was found that when these soaked coupons were tested against S. aureus, complete inhibition (8.1 logs) was obtained. Because organoselenium cannot leach from the polymer, this would imply that the organoselenium polypropylene contact lens case coupon would be inhibitory toward bacterial biofilm for the life of the case. CONCLUSION: The organoselenium polypropylene contact lens case coupon shows the ability to inhibit biofilm formation. The use of organoselenium copolymer should play an important role in protecting against contact lens case-acquired infection.

The Pseudomonas aeruginosa extracellular secondary metabolite, Paerucumarin, chelates iron and is not localized to extracellular membrane vesicles.

Proteins encoded by the Pseudomonas aeruginosa pvcA-D operon synthesize a novel isonitrile functionalized cumarin termed paerucumarin. The pvcA-D operon enhances the expression of the P. aeruginosa fimbrial chaperone/usher pathway (cup) genes and this effect is mediated through paerucumarin. Whether pvcA-D and/or paerucumarin affect the expression of other P. aeruginosa genes is not known. In this study, we examined the effect of a mutation in pvcA-D operon the global transcriptome of the P. aeruginosa strain PAO1-UW. The mutation reduced the expression of several ironcontrolled genes including pvdS, which is essential for the expression of the pyoverdine genes. Additional transcriptional studies showed that the pvcA-D operon is not regulated by iron. Exogenously added paerucumarin enhanced pyoverdine production and pvdS expression in PAO1-UW. Iron-chelation experiments revealed that purified paerucumarin chelates iron. However, exogenously added paerucumarin significantly reduced the growth of a P. aeruginosa mutant defective in pyoverdine and pyochelin production. In contrast to other secondary metabolite, Pseudomonas quinolone signal (PQS), paerucumarin is not localized to the P. aeruginosa membrane vesicles. These results suggest that paerucumarin enhances the expression of iron-controlled genes by chelating iron within the P. aeruginosa extracellular environment. Although paerucumarin chelates iron, it does not function as a siderophore. Unlike PQS, paerucumarin is not associated with the P. aeruginosa cell envelope.

Inhibition of otopathogenic biofilms by organoselenium-coated tympanostomy tubes.

IMPORTANCE: Tube occlusion and post-tympanostomy tube otorrhea (PTTO) are 2 major sequelae of tympanostomy tube placement. Plugging negates the function of the tympanostomy tubes and, along with chronic PTTO, can be financially burdensome owing to repeated surgical procedures and additional treatments. OBJECTIVE: To investigate the effectiveness of an organoselenium (OSe) coating on Donaldson tympanostomy tubes in inhibiting biofilm formation on the tympanostomy tubes. DESIGN: In vitro microbiologic study; all experiments were performed in a Texas Tech University Health Sciences Center basic sciences laboratory. INTERVENTIONS: Inhibition of biofilm formation was investigated by incubating OSe-coated vs uncoated (control) tympanostomy tubes in a nutrient broth containing either Staphylococcus aureus (Sa) expressing green fluorescent protein (GFP), nontypeable Haemophilus influenzae (NTHi) expressing GFP, or Moraxella catarrhalis (Mc) for 48 hours at 37 °C. All biofilms were quantified via colony-forming unit (CFU) assays. The Sa and NTHi biofilms were visualized using confocal laser-scanning microscopy (CLSM) and analyzed using the COMSTAT program. MAIN OUTCOMES AND MEASURES: The CFU assays, CLSM, and COMSTAT analysis revealed that compared with uncoated control tympanostomy tubes, OSe-coated tympanostomy tubes are able to inhibit Sa, NTHi, and Mc biofilm formation. RESULTS: The Sa and NTHi developed thick mature biofilms containing considerable biomass on uncoated tympanostomy tubes as determined by CLSM and COMSTAT analysis, while the OSe coating on the tympanostomy tubes drastically inhibited biofilm formation by Sa and NTHi. Quantitative CFU analysis revealed that this reduction in biofilm formation was significant, 6 logs for Sa (P < .001) and 4 logs for NTHi (P = .02). OSe coating also inhibited biofilm formation by Mc with a 4.5-log reduction (P < .001). CONCLUSIONS AND RELEVANCE: The OSe coating is a potential long-lasting agent to prevent biofilm development on tympanostomy tubes by otopathogens.

Characterization of biofilm-like structures formed by Pseudomonas aeruginosa in a synthetic mucus medium.

BACKGROUND: The accumulation of thick stagnant mucus provides a suitable environment for the growth of Pseudomonas aeruginosa and Staphylococcus aureus within the lung alveoli of cystic fibrosis (CF) patients. These infections cause significant lung damage, leading to respiratory failure and death. In an artificial mucin containing medium ASM+, P. aeruginosa forms structures that resemble typical biofilms but are not attached to any surface. We refer to these structures as biofilm like structures (BLS). Using ASM+ in a static microtiter plate culture system, we examined the roles of mucin, extracellular DNA, environmental oxygen (EO2), and quorum sensing (QS) in the development of biofilm-like structures (BLS) by P. aeruginosa; and the effect of EO2 and P. aeruginosa on S. aureus BLS. RESULTS: Under 20% EO2, P. aeruginosa strain PAO1 produced BLS that resemble typical biofilms but are confined to the ASM+ and not attached to the surface. Levels of mucin and extracellular DNA within the ASM+ were optimized to produce robust well developed BLS. At 10% EO2, PAO1 produced thicker, more developed BLS, while under 0% EO2, BLS production was diminished. In contrast, the S. aureus strain AH133 produced well-developed BLS only under 20% EO2. In PAO1, loss of the QS system genes rhlI and rhlR affected the formation of BLS in ASM+ in terms of both structure and architecture. Whether co-inoculated into ASM+ with AH133, or added to established AH133 BLS, PAO1 eliminated AH133 within 48-56 h. CONCLUSIONS: The thick, viscous ASM+, which contains mucin and extracellular DNA levels similar to those found in the CF lung, supports the formation of biofilm-like structures similar to the aggregates described within CF airways. Alterations in environmental conditions or in the QS genes of P. aeruginosa, as occurs naturally during the progression of CF lung infection, affect the architecture and quantitative structural features of these BLS. Thus, ASM+ provides an in vitro medium in which the effect of changing levels of substances produced by the host and the bacteria can be analyzed to determine the effect on such structures and on the susceptibility of the bacteria within the BLS to various treatments.

Serum albumin alters the expression of iron-controlled genes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, which causes serious infections in immunocompromised patients, produces numerous virulence factors, including exotoxin A and the siderophore pyoverdine. As production of these virulence factors is influenced by the host environment, we examined the effect serum has on global transcription within P. aeruginosa strain PAO1 at different phases of growth in an iron-deficient medium. At early exponential phase, serum significantly enhanced expression of 138 genes, most of which are repressed by iron, including pvdS, regA and the pyoverdine synthesis genes. However, serum did not interfere with the repression of these genes by iron. Serum enhanced regA expression in a fur mutant of PAO1 but not in a pvdS mutant. The serum iron-binding protein apotransferrin, but not ferritin, enhanced regA and pvdS expression. However, in PAO1 grown in a chemically defined medium that contains no iron, serum but not apotransferrin enhanced pvdS and regA expression. While complement inactivation failed to eliminate this effect, albumin absorption reduced the effect of serum on pvdS and regA expression in the iron-deficient medium chelexed tryptic soy broth dialysate. Additionally, albumin absorption eliminated the effect of serum on pvdS and regA expression in the chemically defined medium. These results suggest that serum enhances the expression of P. aeruginosa iron-controlled genes by two mechanisms: one through apotransferrin and another one through albumin.

Regulation of the Pseudomonas aeruginosa toxA, regA and ptxR genes by the iron-starvation sigma factor PvdS under reduced levels of oxygen.

The level of environmental oxygen (EO) within various Pseudomonas aeruginosa infection sites is low (microaerobic), and this can affect the production of different virulence factors. Expression of the toxA gene, encoding exotoxin A (ETA), is regulated by regA, ptxR and pvdS. Moreover, the iron-starvation sigma factor PvdS directs the transcription of pyoverdine siderophore genes (e.g. pvdD). DNA-protein binding analysis using recombinant PvdS showed that the PvdS-RNA polymerase holoenzyme complex specifically bound the toxA, regA and ptxR promoter regions. All three promoters contain a PvdS-binding site, the iron-starvation box. To determine the relationship between these different genes and PvdS, we conducted a comparative analysis of toxA, regA, ptxR and pvdD transcription throughout the growth cycle of wild-type P. aeruginosa and its pvdS mutant in iron-deficient medium under aerobic-shaking (A-sh) and microaerobic-static (M-st) conditions. Under both EO conditions, optimal toxA, regA and pvdD expression and pyoverdine production required PvdS, while ptxR expression was moderately dependent on PvdS only under A-sh conditions. Expression of regA, pvdD and pyoverdine production in wild-type P. aeruginosa was significantly lower under M-st in comparison with A-sh conditions, while the opposite was observed for toxA and ptxR. Although low, the level of toxA expression and ETA production in the pvdS mutant were higher under M-st than under A-sh conditions. Transcription of pvdS and PvdS expression were also reduced by low EO. We propose that the regulation of toxA expression under aerobic conditions primarily involves PvdS, while an additional EO-responsive regulator(s) besides PvdS is required under low EO levels. Thus, PvdS may control the transcription of the ptxR, regA and toxA genes, and respond to EO by acting at different levels of the toxA regulatory cascade.

Effect of static growth and different levels of environmental oxygen on toxA and ptxR expression in the Pseudomonas aeruginosa strain PAO1.

Within certain infection sites, such as the lung of cystic fibrosis patients, Pseudomonas aeruginosa grows statically under either decreased oxygen tension or anaerobic conditions, a situation that is likely to influence the production of virulence factors. The goal of this study was to determine the effect of static growth under microaerobic (decreased oxygen) and anaerobic conditions on the expression of the P. aeruginosa exotoxin A (ETA) gene toxA and its positive regulator ptxR. Using toxA-lacZ and ptxR-lacZ fusion plasmids, the level of toxA and ptxR expression was measured throughout the growth cycle of strain PAO1, which was grown in either iron-deficient or iron-sufficient medium under four different conditions: 20%-SH (aerobic, shaking), 20%-ST (aerobic, static), 10%-ST (microaerobic, static) and 0%-ST (anaerobic, static). In iron-deficient medium, toxA expression was higher under 20%-ST and 10%-ST than under 20%-SH. However, the highest level of toxA expression occurred under 0%-ST. Analysis of ETA protein using sandwich ELISA revealed that at time points between 8 and 24 h of the growth curve, PAO1 produced higher levels of ETA under 0%-ST than under 20%-SH. In iron-sufficient medium, toxA expression was significantly repressed under all conditions. Additional analyses using PAO1 strains that carry lacZ fusions with the toxA regulatory genes regA and pvdS revealed that the expression of regA and pvdS is reduced rather than increased at 0%-ST. ptxR expression under different conditions paralleled that of toxA expression, except that it was repressed by iron under 20 %-SH only. Between 6 and 24 h of growth, and under all conditions, the level of dissolved oxygen (DO) within the PAO1 cultures was sharply reduced. These results suggest that (1) the combined effect of static growth and anaerobic conditions produce a significant increase in toxA and ptxR expression in PAO1; (2) this effect appears to be unique to toxA and ptxR, since the level of regA and pvdS expression was reduced under the same conditions; (3) neither static growth nor anaerobic conditions interfere with the repression of toxA expression by iron, although static growth deregulates ptxR expression with respect to iron; and (4) the enhanced expression of toxA and ptxR is not related to the reduced levels of DO in PAO1 cultures.

Cytokine induction by the P. aeruginosa quorum sensing system during thermal injury.

INTRODUCTION: Pseudomonas aeruginosa causes serious infections in severely burned patients due to its ability to produce numerous virulence factors. The production of most of these factors is controlled by the cell-to-cell communication system called quorum sensing (QS). We have recently shown that several proinflammatory and hematopoietic cytokines are produced during infection of the burn wound with P. aeruginosa strain PAO1. Most of these cytokines were not produced during either thermal injury or P. aeruginosa infection alone. MATERIALS AND METHODS AND RESULTS: In this study, we tried to determine if the QS systems play a role in the production of cytokines during P. aeruginosa infection of burn wounds. This was accomplished using the murine model of thermal injury, the P. aeruginosa strain PAO1 and its QS defective mutant (PAO-JP2), and the Multi-probe RNase protection assay. The mRNA for TNF-alpha, IL-6, TGF-beta, and G-CSF was detected within the skin of PAO1 infected/thermally injured mice. In contrast, the expression of these cytokines was not detected in PAO-JP2 infected/thermally injured mice. In comparison with the parent strain, PAO-JP2 was not defective either in its growth or in its spread within the thermally injured skin. A complementation experiment, using a plasmid that carries the intact QS gene, was conducted to confirm these results. In the presence of the complementing plasmid, PAO-JP2 produced the mRNA for the above cytokines. CONCLUSIONS: These results suggest that: 1) the QS system is involved in the induction of cytokine expression during P. aeruginosa infection of burn wounds; and 2) this effect may be caused by either a component of the QS system or a QS-controlled virulence factor.