The Pneumococcal Iron Uptake Protein a (PiuA) Specifically Recognizes Tetradentate FeIIIbis- and Mono-Catechol Complexes

Streptococcus pneumoniae (Spn) is an important Gram-positive human pathogen that causes millions of infections worldwide with an increasing occurrence of antibiotic resistance. Fe acquisition is a crucial virulence determinant in Spn; further, Spn relies on exogenous FeIII-siderophore scavenging to meet nutritional Fe needs. Recent studies suggest that the human catecholamine stress hormone, norepinephrine (NE), facilitates Fe acquisition in Spn under conditions of transferrin-mediated Fe starvation. Here we show that the solute binding lipoprotein PiuA from the piu Fe acquisition ABC transporter PiuBCDA, previously described as an Fe-hemin binding protein, binds tetradentate catechol FeIII complexes, including NE and the hydrolysis products of enterobactin. Two protein-derived ligands (H238, Y300) create a coordinately-saturated FeIII complex, which parallel recent studies in the Gram-negative intestinal pathogen Campylobacter jejuni. Our in vitro studies using NMR spectroscopy and 54Fe LC-ICP-MS confirm the FeIII can move from transferrin to apo-PiuA in a NE-dependent manner. Structural analysis of PiuA FeIII-bis-catechol and GaIII-bis-catechol and GaIII-(NE)2 complexes by NMR spectroscopy reveals only localized structural perturbations in PiuA upon ligand binding, largely consistent with recent descriptions of other solute binding proteins of type II ABC transporters. We speculate that tetradentate FeIII complexes formed by mono- and bis-catechol species are important Fe sources in Gram-positive human pathogens, since PiuA functions in the same way as SstD from Staphylococcus aureus

Live imaging of the genetically intractable obligate intracellular bacteria orientia tsutsugamushi using a panel of fluorescent dyes

Our understanding of the molecular mechanisms of bacterial infection and pathogenesis are disproportionally derived from a small number of well-characterised species and strains. One reason for this is the enormous time and resources required to develop a new organism into experimental system that can be interrogated at the molecular level, in particular with regards to the development of genetic tools. Live cell imaging by fluorescence microscopy is a powerful technique to study biological processes such as bacterial motility, host cell invasion, and bacterial growth and division. In the absence of genetic tools that enable exogenous expression of fluorescent proteins, fluorescent chemical probes can be used to label and track living cells. A large number of fluorescent chemical probes are commercially available, but these have overwhelmingly been applied to the study of eukaryotic cell systems. Here, we present a methodical analysis of four different classes of probes, which can be used to delineate the cytoplasm, nucleic acids, cell membrane or peptidoglycan of living bacterial cells. We have tested these in the context of the important but neglected human pathogen Orientia tsutsugamushi but expect that the methodology would be broadly applicable to other bacterial species

Live imaging of the genetically intractable obligate intracellular bacteria orientia tsutsugamushi using a panel of fluorescent dyes

Our understanding of the molecular mechanisms of bacterial infection and pathogenesis are disproportionally derived from a small number of well-characterised species and strains. One reason for this is the enormous time and resources required to develop a new organism into experimental system that can be interrogated at the molecular level, in particular with regards to the development of genetic tools. Live cell imaging by fluorescence microscopy is a powerful technique to study biological processes such as bacterial motility, host cell invasion, and bacterial growth and division. In the absence of genetic tools that enable exogenous expression of fluorescent proteins, fluorescent chemical probes can be used to label and track living cells. A large number of fluorescent chemical probes are commercially available, but these have overwhelmingly been applied to the study of eukaryotic cell systems. Here, we present a methodical analysis of four different classes of probes, which can be used to delineate the cytoplasm, nucleic acids, cell membrane or peptidoglycan of living bacterial cells. We have tested these in the context of the important but neglected human pathogen Orientia tsutsugamushi but expect that the methodology would be broadly applicable to other bacterial species

Small molecule detection by reflective interferometric Fourier transform spectroscopy (RIFTS)

A new method for the compensation of matrix effects in biosensing experiments referred to as reflective interferometric Fourier transform spectroscopy (RIFTS) has been developed recently [1]. It employs a porous silicon sensor comprised of two porous silicon layers stacked one on top of the other. The structure has a complicated reflectivity spectrum that can be resolved by FFT analysis leading to three distinctive peaks which are assigned to the layers in the porous silicon structur. If the double layer is appropriately designed, the bottom layer can act as a reference channel. In this paper the specific sensing of small molecules using RIFTS is demonstrated for the first time. Ac-L-Lys-D-Ala-D-Ala has been immobilized to the sensor surface representing the capture probe and vancomycin was used as target analyte

Electroreductive Dimerization of Coumarin and Coumarin Analogues at Carbon Cathodes

Electrochemical reduction of coumarin (<b>1</b>), 6-methylcoumarin (<b>2</b>), 7-methylcoumarin (<b>3</b>), 7-methoxycoumarin (<b>4</b>), and 5,7-dimethoxycoumarin (<b>5</b>) at carbon cathodes in dimethylformamide containing 0.10 M tetra-<i>n</i>-butylammonium tetrafluoroborate has been investigated by means of cyclic voltammetry and controlled-potential (bulk) electrolysis. Cyclic voltammograms for reduction of <b>1</b>–<b>5</b> exhibit two irreversible cathodic peaks: (a) the first peak arises from one-electron reduction of the coumarin to form a radical–anion intermediate, which is protonated by the medium to give a neutral radical; (b) although most of this radical undergoes self-coupling to yield a hydrodimer, reduction of the remaining radical (ultimately to produce a dihydrocoumarin) causes the second cathodic peak. At a potential corresponding to the first voltammetric peak, bulk electrolysis of <b>1</b>–<b>5</b> affords the corresponding hydrodimer as a mixture of <i>meso</i> and <i>dl</i> diastereomers. Although the <i>meso</i> form dominates, the <i>dl</i>-to-<i>meso</i> ratio varies, due to steric effects arising from substituents on the aromatic ring. Electroreduction of an equimolar mixture of <b>1</b> and <b>4</b> gives, along with the anticipated symmetrical hydrodimers, an unsymmetrical product derived from the two coumarins. A mechanistic scheme involving both radical–anion and radical intermediates is proposed to account for the formation of the various products

Host-Polarized Cell Growth in Animal Symbionts

To determine the fundamentals of cell growth, we must extend cell biological studies to non-model organisms. Here, we investigated the growth modes of the only two rods known to widen instead of elongating, Candidatus Thiosymbion oneisti and Thiosymbion hypermnestrae. These bacteria are attached by one pole to the surface of their respective nematode hosts. By incubating live Ca. T. oneisti and T. hypermnestrae with a peptidoglycan metabolic probe, we observed that the insertion of new cell wall starts at the poles and proceeds inward, concomitantly with FtsZ-based membrane constriction. Remarkably, in Ca. T. hypermnestrae, the proximal, animal-attached pole grows before the distal, free pole, indicating that the peptidoglycan synthesis machinery is host oriented. Immunostaining of the symbionts with an antibody against the actin homolog MreB revealed that it was arranged medially—that is, parallel to the cell long axis—throughout the symbiont life cycle. Given that depolymerization of MreB abolished newly synthesized peptidoglycan insertion and impaired divisome assembly, we conclude that MreB function is required for symbiont widening and division. In conclusion, our data invoke a reassessment of the localization and function of the bacterial actin homolog

Unipolar Peptidoglycan Synthesis in the Rhizobiales Requires an Essential Class A Penicillin-Binding Protein

Members of the Rhizobiales are polarly growing bacteria that lack homologs of the canonical Rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant pathogen Agrobacterium tumefaciens. The development of fluorescent d-amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens, enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an additional mode of cell wall synthesis that requires ld-transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella. This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation. IMPORTANCE While the structure and function of the bacterial cell wall are well conserved, the mechanisms responsible for cell wall biosynthesis during elongation are variable. It is increasingly clear that rod-shaped bacteria use a diverse array of growth strategies with distinct spatial zones of cell wall biosynthesis, including lateral elongation, unipolar growth, bipolar elongation, and medial elongation. Yet the vast majority of our understanding regarding bacterial elongation is derived from model organisms exhibiting lateral elongation. Here, we explore the role of penicillin-binding proteins in unipolar elongation of Agrobacterium tumefaciens and related bacteria within the Rhizobiales. Our findings suggest that penicillin-binding protein 1a, along with a subset of ld-transpeptidases, drives unipolar growth. Thus, these enzymes may serve as attractive targets for biocontrol of pathogenic Rhizobiales