Maintenance Of Cell Envelope Integrity Research Articles

Proteomic analysis revealed the survival strategy of Coxiella burnetii to doxycycline exposure

Antibiotic resistance is a global threat with a top concern in healthcare. Doxycycline is an antibiotic highly permeable to cell membrane used for treating a broad variety of bacteria, including Coxiella burnetii. This intracellular pathogen is the causative agent of Q fever, a re-emerging zoonosis found worldwide. Hence, C. burnetii has a considerable impact on the farming industry and public health, it is essential to explore its antibiotic adaptation/tolerance strategy to ensure effective therapy. Herein, we tracked changes in the bacterium induced by doxycycline exposure. Our proteomic analysis detected fifteen significantly altered proteins. Adjustments of some key proteins were verified by gene expression analysis. We also observed an increasing in hydrogen peroxide as a consequence of treatment, indicating deregulation of redox balance. Thus, our data suggests the reduction of protein synthesis to minimal levels, activation of the defense mechanism against oxidative stress and maintenance of cell envelope integrity as the key processes ensuring C. burnetii survival under doxycycline exposure. SignificanceInfection by intracellular microorganisms like C. burnetii requires long periods of treatment, thus antibiotic resistance development is a risk. In this report, 2-DE quantitative proteomics was used to identify changes in the proteome that occurs when C. burnetii is exposed to high concentrations of doxycycline. The identification of pathways impacted by doxycycline could be helpful to understand the mechanism of how C. burnetii is dealing with antibiotic stress.

Extracellular SalB Contributes to Intrinsic Cephalosporin Resistance and Cell Envelope Integrity in Enterococcus faecalis.

Enterococci are major causes of hospital-acquired infections. Intrinsic resistance to cephalosporins is a universal trait among clinically relevant enterococci. Cephalosporin resistance enables enterococci to proliferate to high densities in the intestines of patients undergoing cephalosporin treatment, a precursor to the emergence of infection. However, the genetic and biochemical mechanisms of intrinsic cephalosporin resistance in enterococci are not well understood. A two-component signal transduction system, CroR/S, is required for cephalosporin resistance in enterococci. Although the CroR/S regulon is not well defined, one gene reported to be CroR dependent in Enterococcus faecalis JH2-2 encodes an extracellular putative peptidoglycan hydrolase, SalB. To test the hypothesis that SalB is responsible for CroR-dependent cephalosporin resistance, we examined ΔsalB mutants in multiple genetic lineages of E. faecalis, revealing that SalB is required not only for intrinsic cephalosporin resistance but also for maintenance of cell envelope integrity in the absence of antibiotic stress. The N-terminal signal sequence is necessary for SalB secretion, and secretion is required for SalB to promote cephalosporin resistance. Functional dissection revealed that the C-terminal SCP domain of SalB is essential for biological activity and identified three residues within the SCP domain that are required for the stability and function of SalB. Additionally, we found that in contrast to what is seen in E. faecalis JH2-2, SalB is not regulated by the CroR/S two-component system in E. faecalis OG1, suggesting diversity in the CroR/S regulon among distinct lineages of E. faecalis IMPORTANCE Resistance to cephalosporins is universal among clinically relevant enterococci, enabling enterococcal proliferation to high densities in the intestines of patients undergoing cephalosporin treatment, a precursor to the emergence of infection. Disabling cephalosporin resistance could therefore reduce the incidence of enterococcal infections. However, the genetic and biochemical mechanisms of cephalosporin resistance are not well understood. The significance of this work is the identification of a novel extracellular factor (SalB) that promotes cephalosporin resistance in E. faecalis, which could potentially serve as a target for therapeutics that impair enterococcal cephalosporin resistance. Additionally, our work highlights the importance of the C-terminal SCP domain of SalB, including several conserved residues within the SCP domain, for the ability of SalB to promote cephalosporin resistance.

偏性嫌気性細菌Selenomonas ruminantiumの表層膜安定化機構 : 細胞壁結合型カダベリンの分子機能並びにその合成・制御系

偏性嫌気性細菌Selenomonas ruminantiumの表層膜安定化機構 : 細胞壁結合型カダベリンの分子機能並びにその合成・制御系

Esx Systems and the Mycobacterial Cell Envelope: What's the Connection?

Mycobacterial 6-kDa early secreted antigenic target (ESAT-6) system (ESX) exporters transport proteins across the cytoplasmic membrane. Many proteins transported by ESX systems are then translocated across the mycobacterial cell envelope and secreted from the cell. Although the mechanism underlying protein transport across the mycolate outer membrane remains elusive, the ESX systems are closely connected with and localize to the cell envelope. Links between ESX-associated proteins, cell wall synthesis, and the maintenance of cell envelope integrity have been reported. Genes encoding the ESX systems and those required for biosynthesis of the mycobacterial envelope are coregulated. Here, we review the interplay between ESX systems and the mycobacterial cell envelope.

A tamB homolog is involved in maintenance of cell envelope integrity and stress resistance of Deinococcus radiodurans

The translocation and assembly module (TAM) in bacteria consists of TamA and TamB that form a complex to control the transport and secretion of outer membrane proteins. Herein, we demonstrated that the DR_1462-DR_1461-DR_1460 gene loci on chromosome 1 of Deinococcus radiodurans, which lacks tamA homologs, is a tamB homolog (DR_146T) with two tamB motifs and a DUF490 motif. Mutation of DR_146T resulted in cell envelope peeling and a decrease in resistance to shear stress and osmotic pressure, as well as an increase in oxidative stress resistance, consistent with the phenotype of a surface layer (S-layer) protein SlpA (DR_2577) mutant, demonstrating the involvement of DR_146T in maintenance of cell envelope integrity. The 123 kDa SlpA was absent and only its fragments were present in the cell envelope of DR_146T mutant, suggesting that DR_146T might be involved in maintenance of the S-layer. A mutant lacking the DUF490 motif displayed only a slight alteration in phenotype compared with the wild type, suggesting DUF490 is less important than tamB motif for the function of DR_146T. These findings enhance our understanding of the properties of the multilayered envelope in extremophilic D. radiodurans, as well as the diversity and functions of TAMs in bacteria.

Characterization of the TolB–Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process

The intriguing roles of the bacterial Tol–Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol–Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB–XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB–Pal complex, during the different phases of bacterial biofilm development.

Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division.

To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division.

Multiple roles for Enterococcus faecalis glycosyltransferases in biofilm-associated antibiotic resistance, cell envelope integrity, and conjugative transfer.

The emergence of multidrug-resistant bacteria and the limited availability of new antibiotics are of increasing clinical concern. A compounding factor is the ability of microorganisms to form biofilms (communities of cells encased in a protective extracellular matrix) that are intrinsically resistant to antibiotics. Enterococcus faecalis is an opportunistic pathogen that readily forms biofilms and also has the propensity to acquire resistance determinants via horizontal gene transfer. There is intense interest in the genetic basis for intrinsic and acquired antibiotic resistance in E. faecalis, since clinical isolates exhibiting resistance to multiple antibiotics are not uncommon. We performed a genetic screen using a library of transposon (Tn) mutants to identify E. faecalis biofilm-associated antibiotic resistance determinants. Five Tn mutants formed wild-type biofilms in the absence of antibiotics but produced decreased biofilm biomass in the presence of antibiotic concentrations that were subinhibitory to the parent strain. Genetic determinants responsible for biofilm-associated antibiotic resistance include components of the quorum-sensing system (fsrA, fsrC, and gelE) and two glycosyltransferase (GTF) genes (epaI and epaOX). We also found that the GTFs play additional roles in E. faecalis resistance to detergent and bile salts, maintenance of cell envelope integrity, determination of cell shape, polysaccharide composition, and conjugative transfer of the pheromone-inducible plasmid pCF10. The epaOX gene is located in a variable extended region of the enterococcal polysaccharide antigen (epa) locus. These data illustrate the importance of GTFs in E. faecalis adaptation to diverse growth conditions and suggest new targets for antimicrobial design.

Klebsiella pneumoniae Peptidoglycan-Associated Lipoprotein and Murein Lipoprotein Contribute to Serum Resistance, Antiphagocytosis, and Proinflammatory Cytokine Stimulation

Peptidoglycan-associated lipoprotein (Pal), murein lipoprotein (LppA), and outer membrane protein A (OmpA) are dominant outer membrane proteins (OMPs) that are released by gram-negative bacteria during sepsis. OMPs are implicated in the maintenance of cell envelope integrity. Here, we characterize the roles of these OMPs in pathogenesis during bacteremia caused by Klebsiella pneumoniae. pal-, lppA-, and ompA-deficient K. pneumoniae strains were constructed using an unmarked deletion method. Serum sensitivity, antiphagocytosis activity, outer membrane permeability, and sensitivity to anionic detergents and antimicrobial polypeptides were determined for these OMP gene deletion mutants. The ability of these OMP gene deletion mutants to induce immune responses was compared with that of the wild-type strain in a bacteremic mouse model. Klebsiella pneumoniae strains deleted for pal or lppA exhibited reduced protection from serum killing and phagocytosis; perturbation to the outer membrane permeability barrier and hypersensitivity to bile salts and sodium dodecyl sulfate. The strain mutated for lppA had reduced ability to activate Toll-like receptor 4. Immunization of mice with the pal or lppA mutant provided protection against infection by the wild-type strain. Our findings indicate that K. pneumoniae Pal and LppA proteins are important in the maintenance of cell integrity, contribute to virulence, and could be used as attenuated vaccines.

Comparative proteomic analysis of Lactobacillus plantarumfor the identification of key proteins in bile tolerance

BackgroundLactic acid bacteria are commonly marketed as probiotics based on their putative or proven health-promoting effects. These effects are known to be strain specific but the underlying molecular mechanisms remain poorly understood. Therefore, unravelling the determinants behind probiotic features is of particular interest since it would help select strains that stand the best chance of success in clinical trials. Bile tolerance is one of the most crucial properties as it determines the ability of bacteria to survive in the small intestine, and consequently their capacity to play their functional role as probiotics. In this context, the objective of this study was to investigate the natural protein diversity within the Lactobacillus plantarum species with relation to bile tolerance, using comparative proteomics.ResultsBile tolerance properties of nine L. plantarum strains were studied in vitro. Three of them presenting different bile tolerance levels were selected for comparative proteomic analysis: L. plantarum 299 V (resistant), L. plantarum LC 804 (intermediate) and L. plantarum LC 56 (sensitive). Qualitative and quantitative differences in proteomes were analyzed using two-dimensional electrophoresis (2-DE), tryptic digestion, liquid chromatography-mass spectrometry analysis and database search for protein identification. Among the proteins correlated with differences in the 2-DE patterns of the bacterial strains, 15 have previously been reported to be involved in bile tolerance processes. The effect of a bile exposure on these patterns was investigated, which led to the identification of six proteins that may be key in the bile salt response and adaptation in L. plantarum: two glutathione reductases involved in protection against oxidative injury caused by bile salts, a cyclopropane-fatty-acyl-phospholipid synthase implicated in maintenance of cell envelope integrity, a bile salt hydrolase, an ABC transporter and a F0F1-ATP synthase which participate in the active removal of bile-related stress factors.ConclusionsThese results showed that comparative proteomic analysis can help understand the differential bacterial properties of lactobacilli. In the field of probiotic studies, characteristic proteomic profiles can be identified for individual properties that may serve as bacterial biomarkers for the preliminary selection of strains with the best probiotic potential.

Cell envelope stress response in Gram-positive bacteria

The bacterial cell envelope is the first and major line of defence against threats from the environment. It is an essential and yet vulnerable structure that gives the cell its shape and counteracts the high internal osmotic pressure. It also provides an important sensory interface and molecular sieve, mediating both information flow and the controlled transport of solutes. The cell envelope is also the target for numerous antibiotics. Therefore, the monitoring and maintenance of cell envelope integrity in the presence of envelope perturbating agents and conditions is crucial for survival. The underlying signal transduction is mediated by two regulatory principles, two-component systems and extracytoplasmic function sigma factors, in both the Firmicutes (low-GC) and Actinobacteria (high-GC) branches of Gram-positive bacteria. This study presents a comprehensive overview of cell envelope stress-sensing regulatory systems. This knowledge will then be applied for in-depth comparative genomics analyses to emphasize the distribution and conservation of cell envelope stress-sensing systems. Finally, the cell envelope stress response will be placed in the context of the overall cellular physiology, demonstrating that its regulatory systems are linked not only to other stress responses but also to the overall homeostasis and lifestyle of Gram-positive bacteria.

Involvement of the S-layer proteins Hpi and SlpA in the maintenance of cell envelope integrity in Deinococcus radiodurans R1

The potential functions have been investigated of two proteins in Deinococcus radiodurans R1 predicted to be involved in the maintenance and integrity of the S layer: the hexagonally packed intermediate (Hpi) protein, and SlpA (DR2577), a homologue of an S-layer SlpA protein in Thermus thermophilus. Deletion of the hpi gene had little effect on the structure of the cell envelope or on shear- or solvent-induced stress responses. However, deletion of the slpA gene caused substantial alterations in cell envelope structure, and a significant defect in resistance to solvent and shear stresses compared to the wild-type. Ultrastructural analysis of slpA mutant cells indicated loss of much of the outer Hpi protein carbohydrate coat, the 'pink envelope', and the membrane-like backing layer. Together these results suggest that the SlpA protein may be involved in attachment of the Hpi surface layer to the inner cell envelope, and that SlpA may play an important role in the maintenance of cell envelope integrity in D. radiodurans.

Role of TolR N-terminal, central, and C-terminal domains in dimerization and interaction with TolA and tolQ.

The Tol-PAL system of Escherichia coli is a multiprotein system involved in maintaining the cell envelope integrity and is necessary for the import of some colicins and phage DNA into the bacterium. It is organized into two complexes, one near the outer membrane between TolB and PAL and one in the cytoplasmic membrane between TolA, TolQ, and TolR. In the cytoplasmic membrane, all of the Tol proteins have been shown to interact with each other. Cross-linking experiments have shown that the TolA transmembrane domain interacts with TolQ and TolR. Suppressor mutant analyses have localized the TolQ-TolA interaction to the first transmembrane domain of TolQ and have shown that the third transmembrane domain of TolQ interacts with the transmembrane domain of TolR. To get insights on the composition of the cytoplasmic membrane complex and its possible contacts with the outer membrane complex, we focused our attention on TolR. Cross-linking and immunoprecipitation experiments allowed the identification of Tol proteins interacting with TolR. The interactions of TolR with TolA and TolQ were confirmed, TolR was shown to dimerize, and the resulting dimer was shown to interact with TolQ. Deletion mutants of TolR were constructed, and they allowed us to determine the TolR domains involved in each interaction. The TolR transmembrane domain was shown to be involved in the TolA-TolR and TolQ-TolR interactions, while TolR central and C-terminal domains appeared to be involved in TolR dimerization. The role of the TolR C-terminal domain in the TolA-TolR interaction and its association with the membranes was also demonstrated. Furthermore, phenotypic studies clearly showed that the three TolR domains (N terminal, central, and C terminal) and the level of TolR production are important for colicin A import and for the maintenance of cell envelope integrity.

Expression of the tolQRA genes of Escherichia coli K-12 is controlled by the RcsC sensor protein involved in capsule synthesis.

The tolQRABpal cluster of Escherichia coli K-12 encodes proteins involved in the maintenance of cell-envelope integrity. In addition, tol/pal mutations result in a mucoid colony phenotype at low temperature. The synthesis of capsular polysaccharides by the cps genes is controlled by the positive regulator RcsA and the two-component RcsC/RcsB system. It was shown that the mucoid phenotype of the tol/pal mutants was due to an rcsCB-dependent activation of the cps genes. Furthermore, we have identified a mutation in the rcsC gene that decreased the activity of a tolA-lac operon fusion independently of RcsA and partially independently of RcsB activators. The corresponding rcsC338 mutation resulted in a Glu to Lys substitution at residue 338 of RcsC. This mutation induced mucoidy even at high temperature. We propose that RcsC modulates the phosphorylated forms of RcsB and an uncharacterized regulatory protein involved in the control of the tolQRA genes in an opposite manner. Moreover, our findings strengthen the previous suggestion that RcsC senses some alterations in the cell surface such as those induced by tol, pal or rfa mutations, and activates capsule synthesis to protect the cell against deleterious agents.

Membrane topology of the Escherichia coli TolR protein required for cell envelope integrity

TolR is a 142-amino-acid protein required for the import of colicins and bacteriophage and for maintenance of cell envelope integrity. The topology of TolR in the inner membrane was analyzed by two methods. First, bacteria expressing a series of TolR-beta-galactosidase, TolR-alkaline phosphatase, and TolR-beta-lactamase fusions were assayed for the appropriate enzymatic activity. Second, the accessibility of TolR to proteinase K was determined in permeabilized cells and everted vesicles with an antibody elicited against the carboxyl-terminal 70% of TolR. The results are consistent with TolR spanning the inner membrane once via residues 23 to 43 and with the carboxyl-terminal moiety being exposed to the periplasm. Quantitative studies with the anti-TolR antibody indicated the presence of 2 x 10(3) to 3 x 10(3) TolR molecules per cell.