Engineering a detect and destroy skin probiotic to combat methicillin-resistant Staphylococcus aureus.

The prevalence and virulence of pathogens such as methicillin-resistant Staphylococcus (S.) aureus (MRSA), which can cause recurrent skin infections, are of significant clinical concern. Prolonged antibiotic exposure to treat or decolonize S. aureus contributes to development of antibiotic resistance, as well as depletion of the microbiome, and its numerous beneficial functions. We hypothesized an engineered skin probiotic with the ability to selectively deliver antimicrobials only in the presence of the target organism could provide local bioremediation of pathogen colonization. We constructed a biosensing S. epidermidis capable of detecting the presence of S. aureus quorum sensing autoinducer peptide and producing lysostaphin in response. Here, we demonstrate in vitro activity of this biosensor and present and discuss challenges to deployment of this and other engineered topical skin probiotics

Controlling the Growth of the Skin Commensal Staphylococcus epidermidis Using d-Alanine Auxotrophy.

Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, Staphylococcus epidermidis, for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that requires exogenously supplied d-alanine. The S. epidermidis NRRL B-4268 Δalr1 Δalr2 Δdat strain (SEΔΔΔ) contains deletions of three biosynthetic genes: two alanine racemase genes, alr1 and alr2 (SE1674 and SE1079), and the d-alanine aminotransferase gene, dat (SE1423). These three deletions restricted growth in d-alanine-deficient medium, pooled human blood, and skin. In the presence of d-alanine, SEΔΔΔ colonized and increased expression of human β-defensin 2 in cultured human skin models in vitro. SEΔΔΔ showed a low propensity to revert to d-alanine prototrophy and did not form biofilms on plastic in vitro. These studies support the potential safety and utility of SEΔΔΔ as a live biotherapeutic strain whose growth can be controlled by d-alanine.IMPORTANCE The skin microbiome is rich in opportunities for novel therapeutics for skin diseases, and synthetic biology offers the advantage of providing novel functionality or therapeutic benefit to live biotherapeutic products. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. This study presents the design and in vitro evidence of a skin commensal whose growth can be controlled through d-alanine. The basis of this strain will support future clinical studies of this strain in humans

Tryptophan Scanning Analysis of the Membrane Domain of CTR-Copper Transporters

Membrane proteins of the CTR family mediate cellular copper uptake in all eukaryotic cells and have been shown to participate in uptake of platinum-based anticancer drugs. Despite their importance for life and the clinical treatment of malignancies, directed biochemical studies of CTR proteins have been difficult because high-resolution structural information is missing. Building on our recent 7Å structure of the human copper transporter hCTR1, we present the results of an extensive tryptophan-scanning analysis of hCTR1 and its distant relative, yeast CTR3. The comparative analysis supports our previous assignment of the transmembrane helices and shows that most functionally and structurally important residues are clustered around the threefold axis of CTR trimers or engage in helix packing interactions. The scan also identified residues that may play roles in interactions between CTR trimers and suggested that the first transmembrane helix serves as an adaptor that allows evolutionarily diverse CTRs to adopt the same overall structure. Together with previous biochemical and biophysical data, the results of the tryptophan scan are consistent with a mechanistic model in which copper transport occurs along the center of the trimer

Characterization of Ixophilin, a thrombin inhibitor from the gut of Ixodes scapularis.

Ixodes scapularis, the black-legged tick, vectors several human pathogens including Borrelia burgdorferi, the agent of Lyme disease in North America. Pathogen transmission to the vertebrate host occurs when infected ticks feed on the mammalian host to obtain a blood meal. Efforts to understand how the tick confronts host hemostatic mechanisms and imbibes a fluid blood meal have largely focused on the anticoagulation strategies of tick saliva. The blood meal that enters the tick gut remains in a fluid state for several days during the process of feeding, and the role of the tick gut in maintaining the blood-meal fluid is not understood. We now demonstrate that the tick gut produces a potent inhibitor of thrombin, a key enzyme in the mammalian coagulation cascade. Chromatographic fractionation of engorged tick gut proteins identified one predominant thrombin inhibitory activity associated with an approximately 18 kDa protein, henceforth referred to as Ixophilin. The ixophilin gene was preferentially transcribed in the guts of feeding nymphs. Expression began after 24 hours of feeding, coincident with the flow of host blood into the tick gut. Immunity against Ixophilin delayed tick feeding, and decreased feeding efficiency significantly. Surprisingly, immunity against Ixophilin resulted in increased Borrelia burgdorferi transmission to the host, possibly due to delayed feeding and increased transmission opportunity. These observations illuminate the potential drawbacks of targeting individual tick proteins in a functional suite. They also underscore the need to identify the "anticoagulome" of the tick gut, and to prioritize a critical subset of anticoagulants that could be targeted to efficiently thwart tick feeding, and block pathogen transmission to the vertebrate host

<i>Ixodes scapularis</i> gut extracts inhibit thrombin.

<p><b>A</b>. Inhibition of factor Xa (solid diamonds) and thrombin (solid squares) by an extract from adult tick guts. Each gut was homogenized in approximately 30 µl of PBS. <b>B</b>. Inhibition of factor Xa (solid diamonds) and thrombin (solid squares) by an extract from adult tick salivary glands. Each pair of salivary glands was homogenized in approximately 30 µl of PBS.</p

Ixophilin shows homology with thrombin inhibitors, and is preferentially expressed in the guts of nymphal and adult <i>Ixodes scapularis</i>.

<p><b>A.</b> Annotated sequence of protein encoded by ISCW003862 (Ixophilin). Alternating colors indicate exons, aminoacids underlined in red indicate the end of each exon. Arrow-head indicates the signal cleavage site between Alanine and Glutamine. <b>B.</b> ClustalW alignment of Ixophilin, Boophilin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068012#pone.0068012-MacedoRibeiro1" target="_blank">[17]</a> and Hemalin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068012#pone.0068012-Liao1" target="_blank">[16]</a>. Asterisks (*) indicate identity between all three sequences; colon (:) indicates conservation between groups of strongly similar properties; period (.) indicates conservation between groups of weakly similar properties - scoring = /<0.5 in the Gonnet PAM 250 matrix. <b>C.</b> Quantitative RT-PCR analysis of <i>ixophilin</i> expression in unfed, fed larvae, unfed, 24 h fed, and replete nymph salivary glands (SG) and guts (Gut); and in unfed and replete adult salivary glands (SG) and guts (Gut). Error bars represent mean±SEM of 4 replicates in pools of 5 salivary glands and guts.</p

Primers utilized in this study.

<p>Primers utilized in this study.</p

<i>In vitro</i> efficacy of <i>S</i>. <i>epidermidis</i> producing different antimicrobials.

A) Concept of design to express antimicrobial peptide (AMP) genes under the biosensor. AMPs are tagged for export with a signal peptide (SP) in S. epidermidis under control of either (1) tetracycline inducible promoter (Pxyl/tet) for testing under tight inducible control, or (2) agr P2P3 biosensor. B) Pull-down SDS-PAGE verifying AMPs elafin, LL-37, hiracin, and lysostaphin secretion from S. epidermidis. Several different signaling peptides (e.g., SP-14, SP-15, SP-17) were tested for LL-37. The endogenous signal peptide for lysostaphin was used. C) S. epidermidis expressing AMPs under the tet-inducible promoter demonstrate strong growth inhibition of S. aureus in an overlay assay. Concentration of anhydrotetracycline (ATc) is shown and is dose-dependent. D) In vitro activity and specificity of lysostaphin-expressing agr type I, II, and III biosensors integrated into Tü3298Δagr genome. Parent strain included as control. Two biosensor integrant clones (i.e., biological replicates “1” and “2”) were tested at initial doses of 104 and 106 CFUs of S. epidermidis spotted on for each overlay assay.</p

Primer sets used for amplifying specific DNA fragments by PCR.

Primer sets used for amplifying specific DNA fragments by PCR.</p

Trials to define biosensor control of MRSA proliferation <i>in vivo</i>.

Germ-free C57BL6/J mice were colonized on the ears on days 0, 2 and 4, and growth of S. epidermidis (agr type I lysostaphin producing biosensor or Tü3298Δagr parent strain) and S. aureus was quantified by swabbing the ear, directly spreading on mannitol salt agar, and then counting CFUs on days 2, 4, 6, 8, and 10 (select cases). In A) we show representative control samples for colonization alone by S. aureus USA300 (MRSA), S. epidermidis parent, or S. epidermidis biosensor strains. B) and C), D) and E), and F) and G) are replicate experiments. In B) and C), we examined biosensor efficacy in suppressing growth of MRSA when co-colonized. MRSA was mixed with the biosensor or parent in a suspension at a 1:100 ratio (107/109 CFUs, respectively) and then applied to the mouse ears. n = 5 mice for all groups. Spearman linear regression line is shown with 95% confidence intervals. P-value to assess statistical significance in CFU counts between groups, accounting for time as a covariate, was determined within each panel by ANCOVA. In D) and E), we examined biosensor efficacy in preventing colonization (and suppressing growth), colonizing mouse ears with biosensor or parent alone at day 0, then challenging with the suspension of S. aureus USA300 with S. epidermidis biosensor or parent on day 2. n = 5 for all groups. In F) and G), we examined growth in a wound model, in which mice underwent a dorsal punch biopsy which was then inoculated with the suspension of S. aureus USA300 plus biosensor or parent. After 48 h, dorsal skin surrounding the wound was harvested for CFU quantitation on mannitol salt agar. n = 5 mice/group (left), n = 12/group (right). P-value was determined by bidirectional t-test.</p