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Bacterial adhesion is the initial step in surface colonization and community formation. At the single-cell level, atomic force microscopy (AFM) techniques have enabled the quantification of adhesive forces between bacteria and substrata. However, conventional techniques depend on the irreversible immobilization of cells onto cantilevers, thus hampering throughput. Here, we developed a modular AFM method to reversibly immobilize functionalized beads as surface mimic and to probe adhesion of individual bacteria. We performed single-cell force spectroscopies with phylogenetically diverse leaf isolates of various size and morphology. Adhesion measurement of 28 bacterial strains revealed large differences in hydrophobic interactions of about three orders of magnitude. The highest adhesion forces of up to 50 nN were recorded for members of the Gammaproteobacteria. The hydrophobicity of the different isolates correlated positively with the retention of bacteria observed in planta and might provide a basis for successful leaf colonization and potentially disease outbreaks of pathogens. more...

Rhesus TRIM5α (rhTRIM5α) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5α to the assembled HIV-1 capsid core. This host-pathogen interaction involves multiple capsid molecules within the hexagonal HIV-1 capsid lattice. However, the molecular details of this interaction and the precise site at which the B30.2 domain binds remain largely unknown. The human orthologue of TRIM5α (hsTRIM5α) fails to block infection by HIV-1 both in vivo and in vitro This is thought to be due to differences in binding to the capsid lattice. To map the species-specific binding surface on the HIV-1 capsid lattice, we used microscale thermophoresis and dual-focus fluorescence correlation spectroscopy to measure binding affinity of rhesus and human TRIM5α B30.2 domains to a series of HIV-1 capsid variants that mimic distinct capsid arrangements at each of the symmetry axes of the HIV-1 capsid lattice. These surrogates include previously characterized capsid oligomers, as well as a novel chemically cross-linked capsid trimer that contains cysteine substitutions near the 3-fold axis of symmetry. The results demonstrate that TRIM5α binding involves multiple capsid molecules along the 2-fold and 3-fold interfaces between hexamers and indicate that the binding interface at the 3-fold axis contributes to the well-established differences in restriction potency between TRIM5α orthologues. IMPORTANCE TRIM5α is a cellular protein that fends off infection by retroviruses through binding to the viruses' protein shell surrounding its genetic material. This shell is composed of several hundred capsid proteins arranged in a honeycomb-like hexagonal pattern that is conserved across retroviruses. By binding to the complex lattice formed by multiple capsid proteins, rather than to a single capsid monomer, TRIM5α restriction activity persists despite the high mutation rate in retroviruses such as HIV-1. In rhesus monkeys, but not in humans, TRIM5α confers resistance to HIV-1. By measuring the binding of human and rhesus TRIM5α to a series of engineered HIV-1 capsid mimics of distinct capsid lattice interfaces, we reveal the HIV-1 capsid surface critical for species-specific binding by TRIM5α., (Copyright © 2018 Morger et al.) more...

Bacteriophage-based assays and biosensors rival traditional antibody-based immunoassays for detection of low-level Salmonella contaminations. In this study, we harnessed the binding specificity of the long tail fiber (LTF) from bacteriophage S16 as an affinity molecule for the immobilization, enrichment, and detection of Salmonella We demonstrate that paramagnetic beads (MBs) coated with recombinant gp37-gp38 LTF complexes (LTF-MBs) are highly effective tools for rapid affinity magnetic separation and enrichment of Salmonella Within 45 min, the LTF-MBs consistently captured over 95% of Salmonella enterica serovar Typhimurium cells from suspensions containing from 10 to 10 5 CFU · ml -1 , and they yielded equivalent recovery rates (93% ± 5%, n = 10) for other Salmonella strains tested. LTF-MBs also captured Salmonella cells from various food sample preenrichments, allowing the detection of initial contaminations of 1 to 10 CFU per 25 g or ml. While plating of bead-captured cells allowed ultrasensitive but time-consuming detection, the integration of LTF-based enrichment into a sandwich assay with horseradish peroxidase-conjugated LTF (HRP-LTF) as a detection probe produced a rapid and easy-to-use Salmonella detection assay. The novel enzyme-linked LTF assay (ELLTA) uses HRP-LTF to label bead-captured Salmonella cells for subsequent identification by HRP-catalyzed conversion of chromogenic 3,3',5,5'-tetramethylbenzidine substrate. The color development was proportional for Salmonella concentrations between 10 2 and 10 7 CFU · ml -1 as determined by spectrophotometric quantification. The ELLTA assay took 2 h to complete and detected as few as 10 2 CFU · ml -1 S Typhimurium cells. It positively identified 21 different Salmonella strains, with no cross-reactivity for other bacteria. In conclusion, the phage-based ELLTA represents a rapid, sensitive, and specific diagnostic assay that appears to be superior to other currently available tests. IMPORTANCE The incidence of foodborne diseases has increased over the years, resulting in major global public health issues. Conventional methods for pathogen detection can be laborious and expensive, and they require lengthy preenrichment steps. Rapid enrichment-based diagnostic assays, such as immunomagnetic separation, can reduce detection times while also remaining sensitive and specific. A critical component in these tests is implementing affinity molecules that retain the ability to specifically capture target pathogens over a wide range of in situ applications. The protein complex that forms the distal tip of the bacteriophage S16 long tail fiber is shown here to represent a highly sensitive affinity molecule for the specific enrichment and detection of Salmonella Phage-encoded long tail fibers have huge potential for development as novel affinity molecules for robust and specific diagnostics of a vast spectrum of bacteria., (Copyright © 2017 American Society for Microbiology.) more...