scholarly journals Role of the Cell Wall Microenvironment in Expression of a Heterologous SpaP-S1 Fusion Protein byStreptococcus gordonii

2010 ◽  
Vol 77 (5) ◽  
pp. 1660-1666 ◽  
Author(s):  
Elisabeth Davis ◽  
Dustin Kennedy ◽  
Scott A. Halperin ◽  
Song F. Lee
Keyword(s):  
Cell Wall ◽  
Fusion Protein ◽  
Charge Density ◽  
Mutant Strain ◽  
Cell Envelope ◽  
Regulatory System ◽  
Surface Protein ◽  
Surface Proteins ◽  
Heterologous Proteins ◽  
Two Component

ABSTRACTThe charge density in the cell wall microenvironment of Gram-positive bacteria is believed to influence the expression of heterologous proteins. To test this, the expression of a SpaP-S1 fusion protein, consisting of the surface protein SpaP ofStreptococcus mutansand a pertussis toxin S1 fragment, was studied in the live vaccine candidate bacteriumStreptococcus gordonii. Results showed that the parent strain PM14 expressed very low levels of SpaP-S1. By comparison, thedltmutant strain, which has a mutation in thedltoperon preventingd-alanylation of the cell wall lipoteichoic acids, and another mutant strain, OB219(pPM14), which lacks the LPXTG major surface proteins SspA and SspB, expressed more SpaP-S1 than the parent. Both thedltmutant and the OB219(pPM14) strain had a more negatively charged cell surface than PM14, suggesting that the negative charged cell wall played a role in the increase in SpaP-S1 production. Accordingly, the addition of Ca2+, Mg2+, and K+, presumably increasing the positive charge of the cell wall, led to a reduction in SpaP-S1 production, while the addition of bicarbonate resulted in an increase in SpaP-S1 production. The level of SpaP-S1 production could be correlated with the level of PrsA, a peptidyl-prolylcis/transisomerase, in the cells. PrsA expression appears to be regulated by the cell envelope stress two-component regulatory system LiaSR. The results collectively indicate that the charge density of the cell wall microenvironment can modulate heterologous SpaP-S1 protein expression inS. gordoniiand that this modulation is mediated by the level of PrsA, whose expression is regulated by the LiaSR two-component regulatory system.

scholarly journals A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Ulrike Resch ◽  
James Anthony Tsatsaronis ◽  
Anaïs Le Rhun ◽  
Gerald Stübiger ◽  
Manfred Rohde ◽  
...  
Keyword(s):  
Lipid Composition ◽  
Secretory Pathway ◽  
Group A Streptococcus ◽  
Regulatory System ◽  
Surface Proteins ◽  
Streptococcal Toxic Shock Syndrome ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Two Component ◽  
Group A

ABSTRACT Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS), the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to “anchorless surface proteins.” Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response. IMPORTANCE Group A streptococcus (GAS) is a Gram-positive bacterial pathogen responsible for more than 500,000 deaths annually. Establishment of GAS infection is dependent on a suite of proteins exported via the general secretory pathway. Here, we show that GAS naturally produces extracellular vesicles with a unique lipid composition that are laden with proteins and RNAs. Interestingly, both virulence-associated proteins and RNA species were found to be differentially abundant in vesicles relative to the bacteria. Furthermore, we show that genetic disruption of the virulence-associated two-component regulator CovRS leads to an increase in vesicle production. This study comprehensively describes the protein, RNA, and lipid composition of GAS-secreted MVs and alludes to a regulatory system impacting this process.

scholarly journals Five Genes Encoding Surface-Exposed LPXTG Proteins Are Enriched in Hospital-Adapted Enterococcus faecium Clonal Complex 17 Isolates

2007 ◽  
Vol 189 (22) ◽  
pp. 8321-8332 ◽  
Author(s):  
Antoni P. A. Hendrickx ◽  
Willem J. B. van Wamel ◽  
George Posthuma ◽  
Marc J. M. Bonten ◽  
Rob J. L. Willems
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecium ◽  
Clonal Complex ◽  
Mrna Level ◽  
Surface Protein ◽  
Surface Proteins ◽  
Dot Blot ◽  
Genes Encoding ◽  
Invasive Infections

ABSTRACT Most Enterococcus faecium isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17 E. faecium. Using PCR and Southern and dot blot hybridizations, 131 E. faecium isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the E. faecium TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in E. faecium CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (P < 0.05). Three of these CWAP genes clustered together on the E. faecium TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering E. faecium contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation.

scholarly journals A Novel Sortase, SrtC2, from Streptococcus pyogenes Anchors a Surface Protein Containing a QVPTGV Motif to the Cell Wall

2004 ◽  
Vol 186 (17) ◽  
pp. 5865-5875 ◽  
Author(s):  
Timothy C. Barnett ◽  
Aman R. Patel ◽  
June R. Scott
Keyword(s):  
Cell Wall ◽  
Streptococcus Pyogenes ◽  
Group A Streptococcus ◽  
Surface Protein ◽  
Surface Proteins ◽  
Human Pathogen ◽  
Hydrophobic Region ◽  
C Terminus ◽  
Group A ◽  
Covalently Linked

ABSTRACT The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.

scholarly journals Cell envelope gene expression in phosphate-limited Bacillus subtilis cells

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2470-2484 ◽  
Author(s):  
Eric Botella ◽  
Sebastian Hübner ◽  
Karsten Hokamp ◽  
Annette Hansen ◽  
Paola Bisicchia ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Envelope ◽  
Expression Profiles ◽  
Phosphate Limitation ◽  
Envelope Gene ◽  
Two Component System ◽  
Cell Wall Metabolism ◽  
Component System ◽  
Two Component

The high phosphate content of Bacillus subtilis cell walls dictates that cell wall metabolism is an important feature of the PhoPR-mediated phosphate limitation response. Here we report the expression profiles of cell-envelope-associated and PhoPR regulon genes, determined by live cell array and transcriptome analysis, in exponentially growing and phosphate-limited B. subtilis cells. Control by the WalRK two-component system confers a unique expression profile and high level of promoter activity on the genes of its regulon with yocH and cwlO expression differing both qualitatively and quantitatively from all other autolysin-encoding genes examined. The activity of the PhoPR two-component system is restricted to the phosphate-limited state, being rapidly induced in response to the cognate stimulus, and can be sustained for an extended phosphate limitation period. Constituent promoters of the PhoPR regulon show heterogeneous induction profiles and very high promoter activities. Phosphate-limited cells also show elevated expression of the actin-like protein MreBH and reduced expression of the WapA cell wall protein and WprA cell wall protease indicating that cell wall metabolism in this state is distinct from that of exponentially growing and stationary-phase cells. The PhoPR response is very rapidly deactivated upon removal of the phosphate limitation stimulus with concomitant increased expression of cell wall metabolic genes. Moreover expression of genes encoding enzymes involved in sulphur metabolism is significantly altered in the phosphate-limited state with distinct perturbations being observed in wild-type 168 and AH024 (ΔphoPR) cells.

scholarly journals The BaeSR Two-Component Regulatory System Mediates Resistance to Condensed Tannins in Escherichia coli

2007 ◽  
Vol 74 (2) ◽  
pp. 535-539 ◽  
Author(s):  
Erwin G. Zoetendal ◽  
Alexandra H. Smith ◽  
Monica A. Sundset ◽  
Roderick I. Mackie
Keyword(s):  
Escherichia Coli ◽  
Cell Envelope ◽  
Expression Profiles ◽  
Stress Protein ◽  
Gene Expression Profiles ◽  
Regulatory System ◽  
Multidrug Transporter ◽  
Resistance Strategies ◽  
Envelope Stress ◽  
Two Component

ABSTRACT The gene expression profiles of Escherichia coli strains grown anaerobically with or without Acacia mearnsii (black wattle) extract were compared to identify tannin resistance strategies. The cell envelope stress protein gene spy and the multidrug transporter-encoding operon mdtABCD, both under the control of the BaeSR two-component regulatory system, were significantly up-regulated in the presence of tannins. BaeSR mutants were more tannin sensitive than their wild-type counterparts.

The senX3–regX3 two-component regulatory system of Mycobacterium tuberculosis is required for virulence

Microbiology ◽  
2003 ◽  
Vol 149 (6) ◽  
pp. 1423-1435 ◽  
Author(s):  
Tanya Parish ◽  
Debbie A. Smith ◽  
Gretta Roberts ◽  
Joanna Betts ◽  
Neil G. Stoker
Keyword(s):  
Gene Expression ◽  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Regulatory System ◽  
Normal Growth ◽  
Growth Defect ◽  
Stress Genes ◽  
Two Component ◽  
Whole Genome Microarray ◽  
Immunocompetent Mice

Two-component regulatory systems have been widely implicated in bacterial virulence. To investigate the role of one such system in Mycobacterium tuberculosis, a strain was constructed in which the senX3–regX3 system was deleted by homologous recombination. The mutant strain (Tame15) showed a growth defect after infection of macrophages and was attenuated in both immunodeficient and immunocompetent mice. Competitive hybridization of total RNA from the wild-type and mutant strains to a whole-genome microarray was used to identify changes in gene expression resulting from the deletion. One operon was highly up-regulated in the mutant, indicating that regX3 probably has a role as a repressor of this operon. Other genes which were up- or down-regulated were also identified. Many of the genes showing down-regulation are involved in normal growth of the bacterium, indicating that the mutant strain is subject to some type of growth slow-down or stress. Genes showing differential expression were further grouped according to their pattern of gene expression under other stress conditions. From this analysis 50 genes were identified which are the most likely to be controlled by RegX3. Most of these genes are of unknown function and no obvious motifs were found upstream of the genes identified. Thus, it has been demonstrated that the senX3–regX3 two-component system is involved in the virulence of M. tuberculosis and a number of genes controlled by this system have been identified.

scholarly journals Anchor Structure of Staphylococcal Surface Proteins

2005 ◽  
Vol 280 (16) ◽  
pp. 16263-16271 ◽  
Author(s):  
Luciano A. Marraffini ◽  
Olaf Schneewind
Keyword(s):  
Cell Wall ◽  
Surface Protein ◽  
Surface Proteins ◽  
Sortase A ◽  
Carboxyl Group ◽  
Amino Group ◽  
Sorting Signal ◽  
Lipid Ii ◽  
Sorting Signals ◽  
Anchor Structure

Staphylococcus aureussortase A cleaves surface protein precursors bearing C-terminal LPXTG motif sorting signals between the threonine and glycine residues. Using lipid II precursor as cosubstrate, sortase A catalyzes the amide linkage between the carboxyl group of threonine and the amino group of pentaglycine cross-bridges, thereby tethering C-terminal ends of surface proteins to the bacterial cell wall envelope. Staphylococcal sortase B also anchors its only known substrate, the IsdC precursor with a C-terminal NPQTN motif sorting signal, to the cell wall envelope. Herein, we determined the cell wall anchor structure of IsdC. The sorting signal of IsdC is cleaved between threonine and asparagine of the NPQTN motif, and the carboxyl group of threonine is amide-linked to the amino group of pentaglycine crossbridges. In contrast to sortase A substrates, the anchor structure of IsdC displays shorter glycan strands and significantly less cell wall cross-linking. A model is proposed whereby sortases A and B recognize unique features of sorting signals and peptidoglycan substrates to deposit proteins with distinct topologies in the cell wall envelope.

scholarly journals Loss of Bacterial Cell Pole Stabilization in Caulobacter crescentus Sensitizes to Outer Membrane Stress and Peptidoglycan-Directed Antibiotics

2019 ◽  
Author(s):  
Simon-Ulysse Vallet ◽  
Lykke Haastrup Hansen ◽  
Freja Cecillie Bistrup ◽  
Julien Bortoli Chapalay ◽  
Marc Chambon ◽  
...  
Keyword(s):  
Cell Wall ◽  
Caulobacter Crescentus ◽  
Cell Envelope ◽  
Wall Stress ◽  
Two Component System ◽  
Component System ◽  
Division Plane ◽  
Cell Wall Stress ◽  
Two Component ◽  
Weak Points

AbstractRod-shaped bacteria frequently localise proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the role of such polar factors in responses to extracellular stimuli has been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from Caulobacter crescentus, TipN, and extracellular stress and found that TipN is required for normal tolerance of cell envelope-directed antibiotics, including vancomycin that does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin tolerance. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive ΔtipN strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in ΔtipN cells relative to wild type under some, but not all, cell wall stress conditions that this strain was sensitised to, in particular cefixime and detergent exposure. Together, these results indicate that the ChvIG two-component system has been co-opted as a sensor of cell wall stress and that TipN can influence cell envelope stability and ChvIG-mediated signaling in addition to its roles in intracellular development.Author summaryMaintenance of an intact cell envelope is essential for free-living bacteria to survive harsh conditions they may encounter in their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and re-form parts of the cell envelope at the division plane in order to form new poles as the cells replicate and divide. We have found that TipN, a factor required for correct division and cell pole development in the rod-shaped bacterium, Caulobacter crescentus, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. We also identified an outer membrane receptor, ChvT, that was responsible for allowing vancomycin access to the cells and found that the two-component system that negatively regulates ChvT production was activated by various kinds of cell wall stress. Presence or absence of TipN influenced how active this system was in the presence of cefixime or of the membrane-disrupting detergent sodium deoxycholate. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilisation of these weak points of the cell envelope as well as its other roles inside the cell.

scholarly journals The Two-Component System CesRK Controls the Transcriptional Induction of Cell Envelope-Related Genes in Listeria monocytogenes in Response to Cell Wall-Acting Antibiotics

2008 ◽  
Vol 190 (13) ◽  
pp. 4772-4776 ◽  
Author(s):  
Sanne Gottschalk ◽  
Iver Bygebjerg-Hove ◽  
Mette Bonde ◽  
Pia Kiil Nielsen ◽  
Thanh Ha Nguyen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Envelope ◽  
Two Component System ◽  
Component System ◽  
Two Component ◽  
Transcriptional Induction ◽  
Damaging Effects

ABSTRACT The two-component system CesRK of Listeria monocytogenes responds to cell wall-acting antibiotics. We show here that CesRK controls the transcription of several cell envelope-related genes. The CesRK-dependent induction of these genes may be viewed as an attempt by L. monocytogenes to protect itself against the damaging effects of cell wall-acting antibiotics.

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