The Diguanylate Cyclase HsbD Intersects with the HptB Regulatory Cascade to Control Pseudomonas aeruginosa Biofilm and Motility.

The molecular basis of second messenger signaling relies on an array of proteins that synthesize, degrade or bind the molecule to produce coherent functional outputs. Cyclic di-GMP (c-di-GMP) has emerged as a eubacterial nucleotide second messenger regulating a plethora of key behaviors, like the transition from planktonic cells to biofilm communities. The striking multiplicity of c-di-GMP control modules and regulated cellular functions raised the question of signaling specificity. Are c-di-GMP signaling routes exclusively dependent on a central hub or can they be locally administrated? In this study, we show an example of how c-di-GMP signaling gains output specificity in Pseudomonas aeruginosa. We observed the occurrence in P. aeruginosa of a c-di-GMP synthase gene, hsbD, in the proximity of the hptB and flagellar genes cluster. We show that the HptB pathway controls biofilm formation and motility by involving both HsbD and the anti-anti-sigma factor HsbA. The rewiring of c-di-GMP signaling into the HptB cascade relies on the original interaction between HsbD and HsbA and on the control of HsbD dynamic localization at the cell poles

Correction: The Diguanylate Cyclase HsbD Intersects with the HptB Regulatory Cascade to Control Pseudomonas aeruginosa Biofilm and Motility.

[This corrects the article DOI: 10.1371/journal.pgen.1006354.]

Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing Staphylococcus aureus leukotoxins

Panton-Valentine leukocidin (PVL) is a pore-forming toxin associated with current outbreaks of community-associated methicillin-resistant strains and implicated directly in the pathophysiology of Staphylococcus aureus-related diseases. Humanized heavy chain-only antibodies (HCAb) were generated against S. aureus PVL from immunized transgenic mice to neutralize toxin activity. The active form of PVL consists of the two components, LukS-PV and LukF-PV, which induce osmotic lysis following pore formation in host defense cells. One anti-LukS-PV HCAb, three anti-LukF-PV HCAbs with affinities in the nanomolar range, and one engineered tetravalent bispecific HCAb were tested in vitro and in vivo, and all prevented toxin binding and pore formation. Anti-LukS-PV HCAb also binds to gamma-hemolysin C (HlgC) and inhibits HlgC/HlgB pore formation. Experiments in vivo in a toxin-induced rabbit endophthalmitis model showed that these HCAbs inhibit inflammatory reactions and tissue destruction, with the tetravalent bispecific HCAb performing best. Our findings show the therapeutic potential of HCAbs, and in particular, bispecific antibodies

para-sulfonato-calix[n]arenes inhibit staphylococcal bicomponent leukotoxins by supramolecular interactions.

Panton-Valentine leukocidin (PVL) and other S. aureus β-stranded pore-forming toxins are important virulence factors involved in various but often necrotizing pathologies. This study characterized leukotoxin inhibition by selected para-sulfonato-calix[n]arenes (SCn): SC4, SC6 and SC8. These chemicals have no toxic effects on human erythrocytes or neutrophils, and some are able to inhibit both activity and cell lysis by leukotoxins in a dose-dependent manner. Depending on leukotoxins and SCn, flow cytometry revealed IC50 values between 6-22 µM for Ca2+-activation and between 2-50 µM for cell lysis. SCn were observed to affect membrane binding of class S proteins responsible for cell specificity. Electrospray Mass Spectrometry and Surface Plasmon Resonance established supramolecular interactions (1:1 stoichiometry) between SCn and class S proteins in solution, but not class F proteins. The membrane binding affinity of S proteins ranged from KD = 0.07-6.2 nM. The binding ability was completely abolished by SCn at concentrations according to the number of benzenes (30-300 µM; SC8 < SC6 << SC4). The inhibitory properties of SCn were also observed in vivo in a rabbit model of PVL-induced endophthalmitis. These calixarenes may represent new therapeutic avenues aimed at minimizing inflammatory reactions and necrosis due to certain virulence factors

Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains

Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIEXVIII) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIEXVIII trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIEXVIII modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem VHH-based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas.FJB was supported by a grant from the Ministerio de Economía y Competitividad (CTQ2014-56966-R). JB and BO were supported by a grant from the Ministerio de Economía y Competitividad (BIO2011-22568). AA-C and AB-T were supported by Programa Torres Quevedo from Ministerio de Economía y Competitividad, cofounded by the European Social Fund (PTQ09-01-01089 and PTQ11–04604, respectively). SLH is a recipient of the 2016 Novo Scholarship