High-resolution Visualization of the Microbial Glycocalyx with Low-voltage Scanning Electron Microscopy: Dependence on Cationic Dyes

Erlandsen, Stanley L.; Kristich, Christopher J.; Dunny, Gary M.; Wells, Carol L.

doi:10.1369/jhc.4a6428.2004

Cited by 147 publications

(142 citation statements)

References 29 publications

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“…Introduction of the empty vector did not restore the aggregation phenotype to ⌬SrtA. Immunoblot analysis of fractionated ⌬SrtA cells verified that AS was no longer efficiently incorporated into the cell wall fraction (data not shown) (13).…”

Section: Resultsmentioning

confidence: 92%

Mechanism for Sortase Localization and the Role of Sortase Localization in Efficient Pilus Assembly in Enterococcus faecalis

et al. 2009

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Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.Understanding the transport and processing of proteins in their journey from the cytosol to the extracellular milieu has driven significant advances in elucidating the molecular interactions between an organism and its environment. These interactions are particularly important at the host-pathogen interface, where bacterial adhesins, toxins, and other virulence factors interact with host tissues (31). In gram-negative organisms, transit from the cytosol to the extracellular environment occurs by several mechanisms that either bypass the periplasm or use it as an organelle to process and fold proteins destined for secretion (46). Gram-positive organisms lack a membranebound periplasm but nevertheless secrete many virulence factors that require posttranslational modification (21). It has been proposed that the space between the cell membrane and cell wall provides a protected environment for folding and processing of secreted proteins in gram-positive bacteria (23, 24, 36, 52). Once translocated across the membrane, many virulence factors, such as the Streptococcus pyogenes SpeB protease, are secreted into the extracellular milieu (4), while adhesins are retained at the bacterial surface, where they mediate attachment to host tissues. A large subset of adhesins characterized as virulence factors in gram-positive organisms, such as S. pyogenes M protein and Staphylococcus aureus protein A, are covalently linked to the cell wall by the presence of a cell wall sorting (CWS) signal (1,8,41). The CWS signal is comprised of a C-terminal LPXTG motif, a transmembrane domain, and a positively charged tail (41). Proteins containing this CWS signal are recognized by a sortase enzyme, which cleaves the CWS motif between the threonine-glycine bond. Subsequent transpeptidation links the protein to a lipid II intermediate p...

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Section: Resultsmentioning

confidence: 92%

Mechanism for Sortase Localization and the Role of Sortase Localization in Efficient Pilus Assembly in Enterococcus faecalis

et al. 2009

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“…This RIVET screen and a recent transposon mutagenesis study also undertaken by our group (32) both focused on genetic determinants in the core genome of E. faecalis. While the former study required the use of a microtiter plate biofilm assay, the RIVET study enabled us to use a submerged-coupon system previously shown to support the robust formation of E. faecalis biofilms with distinctive three-dimensional architecture and abundant production of extracellular matrix (18). We suspect that this may account for the fact that many more predicted biofilm determinants were identified in the RIVET screen.…”

Section: Discussionmentioning

confidence: 99%

Functional Genomics ofEnterococcus faecalis: Multiple Novel Genetic Determinants for Biofilm Formation in the Core Genome

et al. 2009

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The ability of Enterococcus faecalis to form robust biofilms on host tissues and on abiotic surfaces such as catheters likely plays a major role in the pathogenesis of opportunistic antibiotic-resistant E. faecalis infections and in the transfer of antibiotic resistance genes. We have carried out a comprehensive analysis of genetic determinants of biofilm formation in the core genome of E. faecalis. Here we describe 68 genetic loci predicted to be involved in biofilm formation that were identified by recombinase in vivo expression technology (RIVET); most of these genes have not been studied previously. Differential expression of a number of these determinants during biofilm growth was confirmed by quantitative reverse transcription-PCR, and genetic complementation studies verified a role in biofilm formation for several candidate genes. Of particular interest was genetic locus EF1809, predicted to encode a regulatory protein of the GntR family. We isolated 14 independent nonsibling clones containing the putative promoter region for this gene in the RIVET screen; EF1809 also showed the largest increase in expression during biofilm growth of any of the genes tested. Since an in-frame deletion of EF1809 resulted in a severe biofilm defect that could be complemented by the cloned wild-type gene, we have designated EF1809 ebrA (enterococcal biofilm regulator). Most of the novel genetic loci identified in our studies are highly conserved in gram-positive bacterial pathogens and may thus constitute a pool of uncharacterized genes involved in biofilm formation that may be useful targets for drug discovery.

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“…The high polysaccharide content of the endothelial glycocalyx interacts poorly with the commonly used postfixation stains. As a result, the endothelial glycocalyx scatters few electrons and is indistinguishable from its environment in conventionally processed samples for transmission electron microscopy (TEM) (24). Thus, even if the pretreatment of the sample maintains endothelial glycocalyx integrity, an additional staining or preservation is required for the visualization of the glycocalyx in TEM.…”

Section: Studying the Endothelial Glycocalyxmentioning

confidence: 99%

A microscopic view on the renal endothelial glycocalyx

Dane

Berg

Lee

et al. 2015

American Journal of Physiology-Renal Physiology

106

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High-resolution Visualization of the Microbial Glycocalyx with Low-voltage Scanning Electron Microscopy: Dependence on Cationic Dyes

Cited by 147 publications

References 29 publications

Mechanism for Sortase Localization and the Role of Sortase Localization in Efficient Pilus Assembly in Enterococcus faecalis

Mechanism for Sortase Localization and the Role of Sortase Localization in Efficient Pilus Assembly in Enterococcus faecalis

Functional Genomics ofEnterococcus faecalis: Multiple Novel Genetic Determinants for Biofilm Formation in the Core Genome

A microscopic view on the renal endothelial glycocalyx

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