Cerium Nitrate Stiffens In Vitro Skin Models and Reduces Pseudomonas aeruginosa Pathogenicity and Penetration Through Skin Models.

Objective: Cerium nitrate (CeN) plus silver sulfadiazine (SSD) cream has been used for 40-plus years to manage burns. CeN produces a hardened eschar believed to resist bacterial colonization/infection. To evaluate this potential mechanism, we treated in vitro skin models or Pseudomonas aeruginosa with CeN and measured mechanical properties of the models and bacterial virulence, respectively. Approach: We treated three-dimensional-collagen matrix and ex-vivo-burned porcine skin with CeN and evaluated stiffness and P. aeruginosa penetration. In addition, we treated P. aeruginosa with CeN and evaluated the bacteria's motility, skin model penetration, susceptibility to be phagocytized by the human monocytic cell line THP-1, and ability to stimulate this cell line to produce cytokines. Results: CeN treatment of skin models stiffened them and made them resistant to P. aeruginosa penetration. Inversely, CeN treatment of P. aeruginosa reduced their motility, penetration through skin models (ex-vivo-burned porcine skin), and ability to stimulate cytokine production (tumor necrosis factor-α [TNF-α] and interleukin 8 [IL-8]) by THP-1 cells. In addition, CeN-treated Pseudomonas was more readily phagocytized by THP-1 cells. Finally, P. aeruginosa inoculated on CeN-treated ex-vivo-burned porcine skin was more susceptible to killing by a silver dressing. Innovation: In vitro skin models offer a platform for screening drugs that interfere with bacterial penetration into wounded tissue. Conclusion: CeN treatment reduced P. aeruginosa virulence, altered the mechanical properties of ex-vivo-burned porcine skin and collagen matrix, retarded penetration of P. aeruginosa through the skin models, and resulted in increased vulnerability of P. aeruginosa to killing by antimicrobial wound dressings. These data support the use of CeN in burn management.

Pseudomonas aeruginosa transcriptome adaptations from colonization to biofilm infection of skin wounds.

In burn patients Pseudomonas aeruginosa infection is a major cause of morbidity. Analysis of the pathogen's gene expression as it transitions from colonization to acute and then biofilm wound infection may provide strategies for infection control. Toward this goal, we seeded log-phase P. aeruginosa (PAO1) into 3-day-old, full-thickness excision wounds (rabbit ear) and harvested the bacteria during colonization (Hrs 2 and 6), acute infection (Hr 24), and biofilm infection (Days 5 and 9) for transcriptome analysis (RNA-Seq). After 2-6 h in the wound, genes for metabolism and cell replication were down-regulated while wound-adaptation genes were up-regulated (vs. expression in log-phase culture). As the infection progressed from acute to biofilm infection, more genes became up-regulated than down-regulated, but the down-regulated genes enriched in more pathways, likely because the genes and pathways that bacteria already colonizing wounds up-regulate to establish biofilm infection are less known. Across the stages of infection, carbon-utilization pathways shifted. During acute infection, itaconate produced by myeloid cells appears to have been a carbon source because myeloid cell infiltration and the expression of the host gene, ACOD1, for itaconate production peaked coincidently with the expression of the PAO1 genes for itaconate transport and catabolism. Additionally, branched-chain amino acids are suggested to be a carbon source in acute infection and in biofilm infection. In biofilm infection, fatty acid degradation was also up-regulated. These carbon sources feed into the glyoxylate cycle that was coincidently up-regulated, suggesting it provided the precursors for P. aeruginosa to synthesize macromolecules in establishing wound infection.

The impact of simultaneous inoculation of Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans on rodent burn wounds.

Burn wound infection often involves a diverse combination of bacterial and fungal pathogens. In this study, we characterize the mixed species burn wound infection by inoculating the burn surface with 1 × 103/4/5 CFU of Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans in a 1:1:1 ratio. Using the revised Walker-Mason scald burn rat model, 168 male Sprague-Dawley rats (350-450 g) subject to ∼10% TBSA burn injury, with or without inoculation, were evaluated for 11 days after burn. In the wound, P. aeruginosa and S. aureus formed robust biofilms as determined by the bacterial tissue load, ∼1 × 109 CFU/g, and expression of key biofilm genes. Interestingly, within 3 days C. albicans achieved tissue loads of ∼1 × 106 CFU/g, but its numbers were significantly reduced beyond the limit of detection in the burn wound by day 7 in partial-thickness injuries and by day 11 in full-thickness injuries. The pathogenic biofilms contributed to burn depth progression, increased release of HMGB-1 into circulation from injured tissue, and significantly elevated the numbers of circulating innate immune cells (Neutrophils, Monocytes, and Basophils). This robust model of multi-species burn wound infection will serve as the basis for the development of new antimicrobials for combating biofilm-based wound infections.

Pirfenidone regulates LPS mediated activation of neutrophils.

Excessive inflammation or its absence may result in impaired wound healing. Neutrophils are among the first innate immune cells to arrive at the injury site. They participate in infection control and debris removal to initiate healing. If not timely resolved, neutrophils can cause excessive tissue inflammation and damage. Drugs with anti-inflammatory and anti-fibrotic effects are of promise for improving healing by balancing the primary defensive functions and excessive tissue damage actions. Of interest, pirfenidone (Pf), an FDA approved anti-fibrotic drug to treat idiopathic pulmonary fibrosis, has been shown to ameliorate inflammation in several animal models including mouse deep partial-thickness burn wounds. However, there is a lack of mechanistic insights into Pf drug action on inflammatory cells such as neutrophils. Here, we examined the treatment effects of Pf on LPS-stimulated neutrophils as a model of non-sterile inflammation. Firstly, Pf reduced chemotaxis and production of pro-inflammatory ROS, cytokines, and chemokines by LPS-activated neutrophils. Secondly, Pf increased anti-inflammatory IL-1RA and reduced neutrophil degranulation, phagocytosis, and NETosis. Thirdly, Pf affected downstream signaling kinases which might directly or indirectly influence neutrophil responses to LPS. In conclusion, the results suggest that Pf lessens the inflammatory phenotypes of LPS-activated neutrophils.

T3SS and alginate biosynthesis of Pseudomonas aeruginosa impair healing of infected rabbit wounds.

Pseudomonas aeruginosa (a Gram-negative bacterium) is an opportunistic pathogen found in many infected wounds and is known to impair healing. To test the hypothesis that knocking out P. aeruginosa genes that are overexpressed during wound infection can cripple a pathogen's ability to impair healing, we assessed two pathways: the Type III secretion system (T3SS) and alginate biosynthesis. We generated single- and double-mutant strains of ExsA (T3SS activator), AlgD (GDP- mannose 6-dehydrogenase of alginate biosynthesis) and their complemented strains and evaluated their pathogenicity in a rabbit ear full-thickness excision-wound infection model. Wounds were inoculated with different strains (wild type, mutants, and complementary strains) at 106 CFU/wound on post-wounding day 3. After 24 h, 5 days and 9 days post-infection, wounds were harvested for measuring bacterial counts (viable and total) and wound healing (epithelial gap). On day 9 post-infection, the viable counts of the double mutant, (exsA/algD)‾ were 100-fold lower than the counts of the wild type (PAO1), single mutants, or the complement double-mutant, (exsA/algD)‾/+. Also, when compared to wounds infected with wild type or control strains, wounds infected with the double-knockout mutant was less inhibitory to wound healing (p < 0.05). Additionally, the double mutant showed greater susceptibility to macrophage phagocytosis in vitro than all other strains (p < 0.001). In conclusion, compared to single gene knockouts, double knockout of virulence genes in T3SS pathway and alginate biosynthesis pathway is more effective in reducing P. aeruginosa pathogenicity and its ability to impair wound healing. This study highlights the necessity of a dual-targeted anti-virulence strategy to improve healing outcomes of P. aeruginosa-infected wounds.

Formation of Pseudomonas aeruginosa Biofilms in Full-thickness Scald Burn Wounds in Rats.

Using Sprague-Dawley rats (350-450 g; n = 61) and the recently updated Walker-Mason rat scald burn model, we demonstrated that Pseudomonas aeruginosa readily formed biofilms within full-thickness burn wounds. Following the burn, wounds were surface-inoculated with P. aeruginosa in phosphate-buffered saline (PBS), while sterile PBS was used for controls. On post-burn days 1, 3, 7, and 11, animals were euthanized and samples collected for quantitative bacteriology, bacterial gene expression, complete blood cell counts, histology, and myeloperoxidase activity. Robust biofilm infections developed in the full-thickness burn wounds inoculated with 1 × 104 CFU of P. aeruginosa. Both histology and scanning electron microscopy showed the pathogen throughout the histologic cross-sections of burned skin. Quantigene analysis revealed significant upregulation of alginate and pellicle biofilm matrix genes of P. aeruginosa within the burn eschar. Additionally, expression of P. aeruginosa proteases and siderophores increased significantly in the burn wound environment. Interestingly, the host's neutrophil response to the pathogen was not elevated in either the eschar or circulating blood when compared to the control burn. This new full-thickness burn biofilm infection model will be used to test new anti-biofilm therapies that may be deployed with soldiers in combat for immediate use at the site of burn injury on the battlefield.

Development of Pseudomonas aeruginosa Biofilms in Partial-Thickness Burn Wounds Using a Sprague-Dawley Rat Model.

We used a modified Walker-Mason scald burn rat model to demonstrate that Pseudomonas aeruginosa, a common opportunistic pathogen in the burn ward and notable biofilm former, establishes biofilms within deep partial-thickness burn wounds in rats.Deep partial-thickness burn wounds, ~10% of the TBSA, were created in anesthetized male Sprague-Dawley rats (350-450 g; n = 84). Immediately post-burn, 100 µl of P. aeruginosa in phosphate-buffered saline at 1 × 103, 1 × 104, or 1 × 105 cells/wound was spread over the burn surface . At 1, 3, 7, and 11 days post-burn, animals were euthanized and blood and tissue were collected for complete blood counts, colony-forming unit (CFU) counts, biofilm gene expression, histology, scanning electron microscopy (SEM), and myeloperoxidase activity in the burn eschar.P. aeruginosa developed robust biofilm wound infections, plateauing at ~1 × 109 CFU/g burn tissue within 7 days regardless of inoculum size. Expression of Pseudomonas alginate genes and other virulence factors in the infected wound indicated formation of mature P. aeruginosa biofilm within the burn eschar. Compared to un-inoculated wounds, P. aeruginosa infection caused both local and systemic immune responses demonstrated by changes in systemic neutrophil counts, histology, and myeloperoxidase activity within the burn wound. Additionally, SEM showed P. aeruginosa enmeshed within an extracellular matrix on the burn surface as well as penetrating 500-600 µm deep into the eschar.P. aeruginosa establishes biofilms within deep partial-thickness burn wounds and invades deep into the burned tissue. This new in vivo biofilm infection model is valuable for testing novel anti-biofilm agents to advance burn care.

Short-Chain Fatty Acids Alter Metabolic and Virulence Attributes of Borrelia burgdorferi.

Borrelia burgdorferi responds to a variety of host-derived factors and appropriately alters its gene expression for adaptation under different host-specific conditions. We previously showed that various levels of acetate, a short-chain fatty acid (SCFA), altered the protein profile of B. burgdorferi In this study, we determined the effects of other physiologically relevant SCFAs in the regulation of metabolic/virulence-associated proteins using mutant borrelial strains. No apparent increase in the synthesis of outer surface protein C (OspC) was noted when a carbon storage regulator A (csrA of B. burgdorferi, or csrABb ) mutant (mt) was propagated within dialysis membrane chambers implanted within rat peritoneal cavity, while the parental wild type (wt; B31-A3 strain) and csrABb cis-complemented strain (ct) had increased OspC with a reciprocal reduction in OspA levels. Growth rates of wt, mt, ct, 7D (csrABb mutant lacking 7 amino acids at the C terminus), and 8S (csrABb with site-specific changes altering its RNA-binding properties) borrelial strains were similar in the presence of acetate. Increased levels of propionate and butyrate reduced the growth rates of all strains tested, with mt and 8S exhibiting profound growth deficits at higher concentrations of propionate. Transcriptional levels of rpoS and ospC were elevated on supplementation of SCFAs compared to those of untreated spirochetes. Immunoblot analysis revealed elevated levels of RpoS, OspC, and DbpA with increased levels of SCFAs. Physiological levels of SCFAs prevalent in select human and rodent fluids were synergistic with mammalian host temperature and pH to increase the levels of aforementioned proteins, which could impact the colonization of B. burgdorferi during the mammalian phase of infection.

Creation of deletion and insertion clonal complex 8 Staphylococcus aureus mutants using a common cloning vector.

Deletion and insertion clonal complex 8 Staphylococcus aureus mutants were created without using intermediate host S. aureus RN4220 or temperature-sensitive shuttle vectors. These mutants were created using a common cloning vector by passing the constructs through a modification host and recovering the electroporated cells in a large volume of medium.

Analysis of DNA and RNA Binding Properties of Borrelia burgdorferi Regulatory Proteins.

Bioinformatic approaches and a large volume of prokaryotic genome sequences have enabled rapid identification of regulatory proteins with features to bind DNA or RNA in a given prokaryote. However, biological relevance of these regulatory proteins requires methods to rapidly purify and determine their binding properties within the physiological context or life style of the organism. Here, we describe the experimental approaches to determine the nucleic acid binding properties of regulatory proteins of Borrelia burgdorferi using Borrelia host-adaptation Re.3gulator (BadR-a DNA binding protein) and Carbon storage regulators A of B. b urgdorferi (CsrABb-an RNA binding protein) as examples. Best laboratory practices associated with overexpression/purification of recombinant borrelial proteins, synthesis of target nucleic acid sequences, and electrophoretic mobility assays to assess the protein/nucleic acid interactions are described. The methods described are intended to facilitate empirical assessment of the binding affinity, co-factor requirements, quality of the interacting partners, and readily modifiable assay conditions to assess the binding properties to define known and unknown regulatory properties of nucleic acid binding proteins of B. burgdorferi.

Spermine and Spermidine Alter Gene Expression and Antigenic Profile of Borrelia burgdorferi.

Borrelia burgdorferi, the agent of Lyme disease, responds to numerous host-derived signals to alter adaptive capabilities during its enzootic cycle in an arthropod vector and mammalian host. Molecular mechanisms that enable B. burgdorferi to detect, channel, and respond to these signals have become an intense area of study for developing strategies to limit transmission/infection. Bioinformatic analysis of the borrelial genome revealed the presence of polyamine transport components (PotA, PotB, PotC, and PotD), while homologs for polyamine biosynthesis were conspicuously absent. Although potABCD is cotranscribed, the level of PotA was elevated under in vitro growth conditions mimicking unfed ticks compared to the level in fed ticks, while the levels of PotD were similar under the aforementioned conditions in B. burgdorferi Among several polyamines and polyamine precursors, supplementation of spermine or spermidine in the borrelial growth medium induced synthesis of major regulators of gene expression in B. burgdorferi, such as RpoS and BosR, with a concomitant increase in proteins that contribute to colonization and survival of B. burgdorferi in the mammalian host. Short transcripts of rpoS were elevated in response to spermidine, which was correlated with increased protein levels of RpoS. Transcriptional analysis of rpoZ and B. burgdorferirel (relBbu ; bb0198) in the presence of spermidine revealed the interplay of multiple regulatory factors in B. burgdorferi gene expression. The effect of spermidine on the levels of select borrelial proteins was also influenced by serum factors. These studies suggest that multiple host-derived signals/nutrients and their transport systems contribute to B. burgdorferi adaptation during the vector and vertebrate host phases of infection.

Global transcriptome responses including small RNAs during mixed-species interactions with methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

Pseudomonas aeruginosa and Staphylococcus aureus mixed-species biofilm infections are more resilient to biocide attacks compared to their single-species counterparts. Therefore, this study used an in vitro model recapitulating bacterial burdens seen in in vivo infections to investigate the interactions of P. aeruginosa and S. aureus in biofilms. RNA sequencing (RNA-seq) was utilized to identify the entire genomic response, both open reading frames (ORFs) and small RNAs (sRNAs), of each species. Using competitive indexes, transposon mutants validated uncharacterized PA1595 of P. aeruginosa and Panton-Valentine leukocidin ORFs of S. aureus are required for competitive success. Assessing spent media on biofilm development determined that the effects of these ORFs are not solely mediated by mechanisms of secretion. Unlike PA1595, leukocidin (lukS-PV) mutants of S. aureus lack a competitive advantage through contact-mediated mechanisms demonstrated by cross-hatch assays. RNA-seq results suggested that during planktonic mixed-species growth there is a robust genomic response or active combat from both pathogens until a state of equilibrium is reached during the maturation of a biofilm. In mixed-species biofilms, P. aeruginosa differentially expressed only 0.3% of its genome, with most ORFs necessary for growth and biofilm development, whereas S. aureus modulated approximately 5% of its genome, with ORFs suggestive of a phenotype of increased virulence and metabolic quiescence. Specific expression of characterized sRNAs aligned with the genomic response to presumably coordinate the adaptive changes necessary for this homeostatic mixed-species biofilm and sRNAs may provide viable foci for the design of future therapeutics.

RNA-Seq Transcriptomic Responses of Full-Thickness Dermal Excision Wounds to Pseudomonas aeruginosa Acute and Biofilm Infection.

Pseudomonas aeruginosa infections of wounds in clinical settings are major complications whose outcomes are influenced by host responses that are not completely understood. Herein we evaluated transcriptomic changes of wounds as they counter P. aeruginosa infection-first active infection, and then chronic biofilm infection. We used the dermal full-thickness, rabbit ear excisional wound model. We studied the wound response: towards acute infection at 2, 6, and 24 hrs after inoculating 106 bacteria into day-3 wounds; and, towards more chronic biofilm infection of wounds similarly infected for 24 hrs but then treated with topical antibiotic to coerce biofilm growth and evaluated at day 5 and 9 post-infection. The wounds were analyzed for bacterial counts, expression of P. aeruginosa virulence and biofilm-synthesis genes, biofilm morphology, infiltrating immune cells, re-epithelialization, and genome-wide gene expression (RNA-Seq transcriptome). This analysis revealed that 2 hrs after bacterial inoculation into day-3 wounds, the down-regulated genes (infected vs. non-infected) of the wound edge were nearly all non-coding RNAs (ncRNAs), comprised of snoRNA, miRNA, and RNU6 pseudogenes, and their down-regulation preceded a general down-regulation of skin-enriched coding gene expression. As the active infection intensified, ncRNAs remained overrepresented among down-regulated genes; however, at 6 and 24 hrs they changed to a different set, which overlapped between these times, and excluded RNU6 pseudogenes but included snRNA components of the major and minor spliceosomes. Additionally, the raw counts of multiple types of differentially-expressed ncRNAs increased on post-wounding day 3 in control wounds, but infection suppressed this increase. After 5 and 9 days, these ncRNA counts in control wounds decreased, whereas they increased in the infected, healing-impaired wounds. These data suggest a sequential and coordinated change in the levels of transcripts of multiple major classes of ncRNAs in wound cells transitioning from inflammation to the proliferation phase of healing.

RsmW, Pseudomonas aeruginosa small non-coding RsmA-binding RNA upregulated in biofilm versus planktonic growth conditions.

BACKGROUND: Biofilm development, specifically the fundamentally adaptive switch from acute to chronic infection phenotypes, requires global regulators and small non-coding regulatory RNAs (sRNAs). This work utilized RNA-sequencing (RNA-seq) to detect sRNAs differentially expressed in Pseudomonas aeruginosa biofilm versus planktonic state. RESULTS: A computational algorithm was devised to detect and categorize sRNAs into 5 types: intergenic, intragenic, 5'-UTR, 3'-UTR, and antisense. Here we report a novel RsmY/RsmZ-type sRNA, termed RsmW, in P. aeruginosa up-transcribed in biofilm versus planktonic growth. RNA-Seq, 5'-RACE and Mfold predictions suggest RsmW has a secondary structure with 3 of 7 GGA motifs located on outer stem loops. Northern blot revealed two RsmW binding bands of 400 and 120 bases, suggesting RsmW is derived from the 3'-UTR of the upstream hypothetical gene, PA4570. RsmW expression is elevated in late stationary versus logarithmic growth phase in PB minimal media, at higher temperatures (37 °C versus 28 °C), and in both gacA and rhlR transposon mutants versus wild-type. RsmW specifically binds to RsmA protein in vitro and restores biofilm production and reduces swarming in an rsmY/rsmZ double mutant. PA4570 weakly resembles an RsmA/RsmN homolog having 49 % and 51 % similarity, and 16 % and 17 % identity to RsmA and RsmN amino acid sequences, respectively. PA4570 was unable to restore biofilm and swarming phenotypes in ΔrsmA deficient strains. CONCLUSION: Collectively, our study reveals an interesting theme regarding another sRNA regulator of the Rsm system and further unravels the complexities regulating adaptive responses for Pseudomonas species.

Whole-Genome Sequence of Multidrug-Resistant Pseudomonas aeruginosa Strain BAMCPA07-48, Isolated from a Combat Injury Wound.

We report here the complete genome sequence of Pseudomonas aeruginosa strain BAMCPA07-48, isolated from a combat injury wound. The closed genome sequence of this isolate is a valuable resource for pathogenome characterization of P. aeruginosa associated with wounds, which will aid in the development of a higher-resolution phylogenomic framework for molecular-guided pathogen-surveillance.

Genome Sequence of a Virulent Pseudomonas aeruginosa Strain, 12-4-4(59), Isolated from the Blood Culture of a Burn Patient.

Pseudomonas aeruginosa is an opportunistic pathogen that frequently infects wounds, significantly impairs wound healing, and causes morbidity and mortality in burn patients. Here, we report the genome sequence of a virulent strain of P. aeruginosa, 12-4-4(59), isolated from the blood culture of a burn patient.

Contributions of environmental signals and conserved residues to the functions of carbon storage regulator A of Borrelia burgdorferi.

Carbon storage regulator A of Borrelia burgdorferi (CsrABb) contributes to vertebrate host-specific adaptation by modulating activation of the Rrp2-RpoN-RpoS pathway and is critical for infectivity. We hypothesized that the functions of CsrABb are dependent on environmental signals and on select residues. We analyzed the phenotype of csrABb deletion and site-specific mutants to determine the conserved and pathogen-specific attributes of CsrABb. Levels of phosphate acetyltransferase (Pta) involved in conversion of acetyl phosphate to acetyl-coenzyme A (acetyl-CoA) and posttranscriptionally regulated by CsrABb in the csrABb mutant were reduced from or similar to those in the control strains under unfed- or fed-tick conditions, respectively. Increased levels of supplemental acetate restored vertebrate host-responsive determinants in the csrABb mutant to parental levels, indicating that both the levels of CsrABb and the acetyl phosphate and acetyl-CoA balance contribute to the activation of the Rrp2-RpoN-RpoS pathway. Site-specific replacement of 8 key residues of CsrABb (8S) with alanines resulted in increased levels of CsrABb and reduced levels of Pta and acetyl-CoA, while levels of RpoS, BosR, and other members of rpoS regulon were elevated. Truncation of 7 amino acids at the C terminus of CsrABb (7D) resulted in reduced csrABb transcripts and posttranscriptionally reduced levels of FliW located upstream of CsrABb. Electrophoretic mobility shift assays revealed increased binding of 8S mutant protein to the CsrA binding box upstream of pta compared to the parental and 7D truncated protein. Two CsrABb binding sites were also identified upstream of fliW within the flgK coding sequence. These observations reveal conserved and unique functions of CsrABb that regulate adaptive gene expression in B. burgdorferi.

Borrelia host adaptation Regulator (BadR) regulates rpoS to modulate host adaptation and virulence factors in Borrelia burgdorferi.

The RpoS transcription factor of Borrelia burgdorferi is a 'gatekeeper' because it activates genes required for spirochaetes to transition from tick to vertebrate hosts. However, it remains unknown how RpoS becomes repressed to allow the spirochaetes to transition back from the vertebrate host to the tick vector. Here we show that a putative carbohydrate-responsive regulatory protein, designated BadR (Borrelia host adaptation Regulator), is a transcriptional repressor of rpoS. BadR levels are elevated in B. burgdorferi cultures grown under in vitro conditions mimicking unfed-ticks and badR-deficient strains are defective for growth under these same conditions. Microarray and immunoblot analyses of badR-deficient strains showed upregulation of rpoS and other factors important for virulence in vertebrate hosts, as well as downregulation of putative tick-specific determinants (e.g. linear plasmid 28-4 genes). DNA-binding assays revealed BadR binds to upstream regions of rpoS. Site-directed mutations in BadR and the presence of phosphorylated sugars affected BadR's binding to the rpoS promoters. badR-deficient B. burgdorferi were unable to colonize mice. Several putative tick-specific targets have been identified. Our study identified a novel regulator, BadR, and provides a link between nutritional environmental cues utilized by spirochaetes to adaptation to disparate conditions found in the tick and vertebrate hosts.

Effect of levels of acetate on the mevalonate pathway of Borrelia burgdorferi.

Borrelia burgdorferi, the agent of Lyme disease, is a spirochetal pathogen with limited metabolic capabilities that survives under highly disparate host-specific conditions. However, the borrelial genome encodes several proteins of the mevalonate pathway (MP) that utilizes acetyl-CoA as a substrate leading to intermediate metabolites critical for biogenesis of peptidoglycan and post-translational modifications of proteins. In this study, we analyzed the MP and contributions of acetate in modulation of adaptive responses in B. burgdorferi. Reverse-transcription PCR revealed that components of the MP are transcribed as individual open reading frames. Immunoblot analysis using monospecific sera confirmed synthesis of members of the MP in B. burgdorferi. The rate-limiting step of the MP is mediated by HMG-CoA reductase (HMGR) via conversion of HMG-CoA to mevalonate. Recombinant borrelial HMGR exhibited a K(m) value of 132 µM with a V(max) of 1.94 µmol NADPH oxidized minute(-1) (mg protein)(-1) and was inhibited by statins. Total protein lysates from two different infectious, clonal isolates of B. burgdorferi grown under conditions that mimicked fed-ticks (pH 6.8/37°C) exhibited increased levels of HMGR while other members of the MP were elevated under unfed-tick (pH 7.6/23°C) conditions. Increased extra-cellular acetate gave rise to elevated levels of MP proteins along with RpoS, CsrA(Bb) and their respective regulons responsible for mediating vertebrate host-specific adaptation. Both lactone and acid forms of two different statins inhibited growth of B. burgdorferi strain B31, while overexpression of HMGR was able to partially overcome that inhibition. In summary, these studies on MP and contributions of acetate to host-specific adaptation have helped identify potential metabolic targets that can be manipulated to reduce the incidence of Lyme disease.

Oligopeptide permease A5 modulates vertebrate host-specific adaptation of Borrelia burgdorferi.

Borrelia burgdorferi, the agent of Lyme disease, undergoes rapid adaptive gene expression in response to signals unique to its arthropod vector or vertebrate hosts. Among the upregulated genes under vertebrate host conditions is one of the five annotated homologs of oligopeptide permease A (OppA5, BBA34). A mutant lacking oppA5 was constructed in an lp25-deficient isolate of B. burgdorferi strain B31, and the minimal regions of infectivity were restored via a shuttle vector pBBE22 with or without an intact copy of bba34. Immunoblot analysis of the bba34 mutant revealed a reduction in the levels of RpoS, BosR, and CsrA(Bb) with a concomitant reduction in the levels of OspC, DbpA, BBK32, and BBA64. There were no changes in the levels of OspA, NapA, P66, and three other OppA orthologs. Quantitative transcriptional analysis correlated with the changes in the protein levels. However, the bba34 mutant displayed comparable infectivities in the C3H/HeN mice and the wild-type strain, despite the reduction in several pathogenesis-related proteins. Supplementation of the growth medium with increased levels of select components, notably sodium acetate and sodium bicarbonate, restored the levels of several proteins in the bba34 mutant to wild-type levels. We speculate that the transport of acetate appears to contribute to the accumulation of key metabolites, like acetyl phosphate, that facilitate the adaptation of B. burgdorferi to the vertebrate host by the activation of the Rrp2-RpoN-RpoS pathway. These studies underscore the importance of solute transport to host-specific adaptation of B. burgdorferi.