Nucleic Acid Amplification Free-QCM-DNA Biosensor for Burkholderia pseudomallei Detection.

Burkholderia pseudomallei is a gram-negative bacterium that causes the infectious disease melioidosis, a disease that can still be fatal despite appropriate treatment. The bacterium contains the gene clusters for the type III secretion system (TTSS), which are essential for its pathogenicity. This gene was often employed for accurate diagnosis through the laborious process of gene amplification. This work intends to develop a quartz crystal microbalance (QCM)-based TTSS gene detection method without gene amplification approaches to simplify the diagnosis process. In this study, it was demonstrated that a 540 bp sequence flanked by BglI restriction sites within the TTSS1 on the B. pseudomallei genome is an effective target for specific detection of the bacteria. After cultivation and genome extraction, the bacteria can be detected by digesting its genome with BglI in which the TTSS1 fragment is detected by a QCM-DNA biosensor, eliminating the need for nucleic acid amplification. A specific probe designed to bind to the TTSSI fragment was utilized as the receptor on the QCM-DNA biosensor which provided the ability to detect the fragment. The limit of detection of the QCM-DNA biosensor was 0.4 µM of the synthetic DNA target oligonucleotide. The system was also capable of specifically detecting the BglI digested-DNA fragment of B. pseudomallei species with significantly higher signal than B. thailandensis. This study provides evidence for an effective QCM-DNA biosensor that can identify B. pseudomallei without the need for nucleic acid amplification.

3D printed hydrophobic barriers in a paper-based biosensor for point-of-care detection of dengue virus serotypes.

Paper-based biosensor is one of the most commonly used platforms for point-of-care testing (POCT). Among these platforms, microfluidic paper-based analytical devices (µPADs) have the most versatile designs due to the different hydrophobic barrier patterns and layers of the devices. In addition, µPADs can also be used in combination with other biosensor platforms to improve the performance of the device. Simple and convenient methods for fabricating low-cost and design-adjustable hydrophobic barriers on paper are one of the most challenging aspects for creating µPADs. This work demonstrated a simple technique for using the common polylactic acid (PLA) filament and wax filament to create hydrophobic barriers on paper for µPADs using a commercialized 3D printer. As a proof of concept, the papers with 3D printed PLA barrier were used in combination with a fluidic chip in a prototype biosensor, in which the barrier paper housed four cell-free reactions and the fluidic chip achieved sample delivery to the reactions in the device. Our designed prototype was capable of discriminating dengue virus serotypes based on small nucleotide sequence differences. The proposed combination of 3D-printed barrier paper and fluidic chip provides a versatile platform for rapid prototyping of POCT with possible compatibility with various detection systems.

Genetic determinants of Silicibacter sp. TM1040 motility.

Silicibacter sp. TM1040 is a member of the marine Roseobacter clade of Alphaproteobacteria that forms symbioses with unicellular eukaryotic phytoplankton, such as dinoflagellates. The symbiosis is complex and involves a series of steps that physiologically change highly motile bacteria into cells that readily form biofilms on the surface of the host. The initial phases of symbiosis require bacterial motility and chemotaxis that drive the swimming bacteria toward their planktonic host. Cells lacking wild-type motility fail to establish biofilms on host cells and do not produce effective symbioses, emphasizing the importance of understanding the molecular mechanisms controlling flagellar biosynthesis and the biphasic "swim-or-stick" switch. In the present study, we used a combination of bioinformatic and genetic approaches to identify the genes critical for swimming of Silicibacter sp. TM1040. More than 40 open reading frames with homology to known flagellar structural and regulatory genes were identified, most of which are organized into approximately eight operons comprising a 35.4-kb locus, with surprising similarity to the fla2 locus of Rhodobacter sphaeroides. The genome has homologs of CckA, CtrA, FlbT, and FlaF, proteins that in Caulobacter crescentus regulate flagellum biosynthesis. In addition, we uncovered three novel genes, flaB, flaC, and flaD, which encode flagellar regulatory proteins whose functions are likely to involve regulation of motor function (FlaD) and modulation of the swim-or-stick switch (FlaC). The data support the conclusion that Silicibacter sp. TM1040 uses components found in other Alphaproteobacteria, as well as novel molecular mechanisms, to regulate the expression of the genes required for motility and biofilm formation. These unique molecular mechanisms may enhance the symbiosis and survival of Roseobacter clade bacteria in the marine environment.

The ability of Proteus mirabilis to sense surfaces and regulate virulence gene expression involves FliL, a flagellar basal body protein.

Proteus mirabilis is a urinary tract pathogen that differentiates from a short swimmer cell to an elongated, highly flagellated swarmer cell. Swarmer cell differentiation parallels an increased expression of several virulence factors, suggesting that both processes are controlled by the same signal. The molecular nature of this signal is not known but is hypothesized to involve the inhibition of flagellar rotation. In this study, data are presented supporting the idea that conditions inhibiting flagellar rotation induce swarmer cell differentiation and implicating a rotating flagellar filament as critical to the sensing mechanism. Mutations in three genes, fliL, fliF, and fliG, encoding components of the flagellar basal body, result in the inappropriate development of swarmer cells in noninducing liquid media or hyperelongated swarmer cells on agar media. The fliL mutation was studied in detail. FliL- mutants are nonmotile and fail to synthesize flagellin, while complementation of fliL restores wild-type cell elongation but not motility. Overexpression of fliL+ in wild-type cells prevents swarmer cell differentiation and motility, a result also observed when P. mirabilis fliL+ was expressed in Escherichia coli. These results suggest that FliL plays a role in swarmer cell differentiation and implicate FliL as critical to transduction of the signal inducing swarmer cell differentiation and virulence gene expression. In concert with this idea, defects in fliL up-regulate the expression of two virulence genes, zapA and hpmB. These results support the hypothesis that P. mirabilis ascertains its location in the environment or host by assessing the status of its flagellar motors, which in turn control swarmer cell gene expression.

Proteus mirabilis ZapA metalloprotease degrades a broad spectrum of substrates, including antimicrobial peptides.

The 54-kDa extracellular metalloprotease ZapA is an important virulence factor of uropathogenic Proteus mirabilis. While ZapA has the ability to degrade host immunoglobulins (Igs), the dramatic attenuation of virulence in ZapA mutants suggests that this enzyme may have a broader spectrum of activity. This hypothesis was tested by in vitro assays with purified ZapA and an array of purified protein or peptide substrates. The data reveal that many proteins found in the urinary tract are substrates of ZapA proteolysis, including complement (C1q and C3), cell matrix (collagen, fibronectin, and laminin), and cytoskeletal proteins (actin and tubulin). Proteolysis of IgA and IgG was significantly enhanced by conditions that denatured the Igs. It was discovered that the antimicrobial peptides human beta-defensin 1 (hBD1) and LL-37 are readily cleaved by the enzyme. To the best of our knowledge, this is the first report of a bacterial protease capable of cleaving hBD1, a component of the human renal tubule innate immune response. Proteolysis of hBD1 resulted in ca. six peptides, while proteolysis of LL-37 resulted in at least nine products. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of the molecular masses of the reaction products indicated that ZapA preferred no distinct peptide bond. The antimicrobial activity of hBD1 and LL-37 was significantly reduced following ZapA treatment, suggesting that proteolysis results in inactivation of these peptides. The data suggest that a function of ZapA during urinary tract infections is the proteolysis of antimicrobial peptides associated with the innate immune response.