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

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 Km value of 132 µM with a Vmax 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, CsrABb 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

Divulging the Complexities of Deep Partial- and Full-Thickness Burn Wounds Afflicted by Staphylococcus Aureus Biofilms in a Rat Burn Model

Every year, thousands of soldiers and civilians succumb to burn wound trauma with highly unfavorable outcomes. We previously established a modified Walker-Mason rat scald model exhibiting a P. aeruginosa infection. Here we characterize deep partial- (DPT) and full-thickness (FT) burn wounds inoculated with Staphylococcus aureus. Male Sprague-Dawley rats (350–450 g) inflicted with 10% total body surface area burn inoculated with S. aureus (103–5 CFU/wound) were monitored over an 11-day period. S. aureus rapidly dominated the wound bed, with bacterial loads reaching at least 1 × 109 CFU/g tissue in all wounds. Within 3 days, S. aureus biofilm formation occurred based on genetic transcripts and Giemsa staining of the tissue. S. aureus infection resulted in a slightly faster recruitment of neutrophils in FT wounds, which was related to necrotic neutrophils. The extent of the inflammatory response in S. aureus infected burn wounds correlated with elevated G-CSF, GM-CSF, GRO/KC and/or TNF-α levels, but a majority of pro- and anti-inflammatory cytokines (IL-1β, IL-6, IFN-γ, IL-10, and IL-13) were found to be suppressed, compared to burn-only controls. S. aureus infection resulted in dynamic changes in DAMPs, including elevated HMGB-1 and reduced levels of circulating hyaluronan within FT wounds. S. aureus also reduced complement C3 at all time points in DPT and FT wounds. These changes in DAMPs are believed to be correlated with burn severity and S. aureus specific bioburden. Collectively, this model showcases the evasiveness of S. aureus through dampening the immune response to flourish in the burn wound

CsrA Modulates Levels of Lipoproteins and Key Regulators of Gene Expression Critical for Pathogenic Mechanisms of Borrelia burgdorferi▿

Carbon storage regulator A (CsrA) is an RNA binding protein that has been characterized in many bacterial species to play a central regulatory role by modulating several metabolic processes. We recently showed that a homolog of CsrA in Borrelia burgdorferi (CsrABb, BB0184) was upregulated in response to propagation of B. burgdorferi under mammalian host-specific conditions. In order to further delineate the role of CsrABb, we generated a deletion mutant designated ES10 in a linear plasmid 25-negative isolate of B. burgdorferi strain B31 (ML23). The deletion mutant was screened by PCR and Southern blot hybridization, and a lack of synthesis of CsrABb in ES10 was confirmed by immunoblot analysis. Analysis of ES10 propagated at pH 6.8/37°C revealed a significant reduction in the levels of OspC, DbpA, BBK32, and BBA64 compared to those for the parental wild-type strain propagated under these conditions, while there were no significant changes in the levels of either OspA or P66. Moreover, the levels of two regulatory proteins, RpoS and BosR, were also found to be lower in ES10 than in the control strain. Quantitative real-time reverse transcription-PCR analysis of total RNA extracted from the parental strain and csrABb mutant revealed significant differences in gene expression consistent with the changes at the protein level. Neither the csrABb mutant nor the trans-complemented strain was capable of infection following intradermal needle inoculation in C3H/HeN mice at either 103 or 105 spirochetes per mouse. The further characterization of molecular basis of regulation mediated by CsrABb will provide significant insights into the pathophysiology of B. burgdorferi

Oligonucleotides used in this study.

a<p>Restriction enzyme sites are underlined.</p

A model for the role of acetate in modulating the vertebrate host-specific adaptation and the mevalonate pathway in <i>B. burgdorferi</i>.

<p>Intracellular levels of acetate contribute to the activation of the Rrp2-RpoN-RpoS pathway leading to the adaptation of <i>B. burgdorferi</i> to vertebrate host-specific conditions and provide the initiating substrate for the mevalonate pathway. It appears that acetate plays a central role in modulating host-specific adaptation to central metabolic functions critical for survival of <i>B. burgdorferi</i> under different environmental conditions. The rate-limiting step of the mevalonate pathway, HMG-CoA reductase (HMGR), is a potential chemotherapeutic target that can be exploited to reduce the survival of <i>B. burgdorferi</i> and hence a reduction in the incidence of Lyme disease. Sequence analysis of members of the MP in <i>B. burgdorferi</i> indicated significant similarity with ORFs of the MP that have been characterized in <i>L. monocytogenes, P. mevalonii</i> and <i>S. aureus</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038171#pone-0038171-t004" target="_blank">Table 4</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038171#pone.0038171-Fraser1" target="_blank">[16]</a>. Based on this analysis, <i>B. burgdorferi</i> appears to possess all open reading frames (ORFs <i>bb0683</i> to <i>bb0688</i>) necessary for the production of IPP via the mevalonate pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038171#pone.0038171-Fraser1" target="_blank">[16]</a>.</p

Sequence comparison of HMGR homologs.

<p>ClustalX alignment of HMGR from eight different species, representing both Class II (<i>Borrelia burgdorferi, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus</i>) and Class I (Human, Syrian Hamster, <i>Drosophila melanogaster</i>, and <i>Saccharomyces cerevisiae</i>) shows a high degree of similarity with regard to binding sites for HMG-CoA/HMG-CoA reductase inhibitors (ENVIG - boxes) and NAD(P)H (DAMGXN and GTVGG - triangles). Indicated by stars are conserved residues shown to be important for catalysis of HMGR in <i>Pseudomonas mevalonii </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038171#pone.0038171-Friesen1" target="_blank">[29]</a>.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Statins inhibit the enzyme activity of <i>B. burgdorferi</i> HMGR.

<p>(A) Lysates purified HMGR as indicated above the respective lanes were resolved by SDS-12.5%PAGE. Gels were stained with Coomassie blue. Recombinant HMGR of <i>B. burgdorferi</i> purified by FPLC. Lane 1, non-induced <i>E. coli</i> containing pMAL-p2X/<i>hmgr</i>. Lane 2, <i>E. coli</i> containing pMAL-p2X/<i>hmgr</i> induced with 1 mM IPTG. Lane 3, Purified MBP-HMGR fusion protein. (B) Recombinant <i>B. burgdorferi</i> HMGR activity was measures as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038171#s2" target="_blank">Materials and Methods</a>. Statins were added to the enzyme reaction at a concentration of 250 µM. The velocity of the control reaction was calculated as 1.41±0.10 µmol NADPH oxidized minute⁻¹ (mg protein)⁻¹, set as 100% maximal activity in the presence of the statin diluent, DMSO, alone_. Simvastatin lowered HMGR activity to 48.2% of maximal (velocity of 0.87±0.08 µmol NADPH oxidized minute⁻¹ (mg protein)⁻¹), while lovastatin reduced HMGR activity to 61.4% of maximal activity (velocity of 0.68±0.07 µmol NADPH oxidized minute⁻¹ (mg protein)⁻¹). Data shown are the average of three independent assays. Asterisks indicate samples whose values are statistically significantly different between control and treated conditions (**, <i>P</i><0.01).</p