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1.
NPJ Microgravity ; 7(1): 9, 2021 Mar 09.
Article in English | MEDLINE | ID: mdl-33750813

ABSTRACT

Spaceflight uniquely alters the physiology of both human cells and microbial pathogens, stimulating cellular and molecular changes directly relevant to infectious disease. However, the influence of this environment on host-pathogen interactions remains poorly understood. Here we report our results from the STL-IMMUNE study flown aboard Space Shuttle mission STS-131, which investigated multi-omic responses (transcriptomic, proteomic) of human intestinal epithelial cells to infection with Salmonella Typhimurium when both host and pathogen were simultaneously exposed to spaceflight. To our knowledge, this was the first in-flight infection and dual RNA-seq analysis using human cells.

2.
NPJ Microgravity ; 3: 10, 2017.
Article in English | MEDLINE | ID: mdl-28649632

ABSTRACT

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection.

3.
Sci Rep ; 7: 43321, 2017 03 03.
Article in English | MEDLINE | ID: mdl-28256611

ABSTRACT

In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS.


Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Oligopeptides/pharmacology , Pseudomonas aeruginosa/drug effects , Serum/chemistry , A549 Cells , Amikacin/pharmacology , Animals , Bacterial Adhesion/drug effects , Biofilms/growth & development , Cattle , Cell Culture Techniques , Colistin/pharmacology , Drug Synergism , Fetus , Gentamicins/pharmacology , Humans , Lung/drug effects , Lung/microbiology , Lung/ultrastructure , Microbial Sensitivity Tests , Models, Biological , Pseudomonas aeruginosa/growth & development , Tight Junctions/drug effects , Tight Junctions/microbiology , Tight Junctions/ultrastructure , Tobramycin/pharmacology
4.
PLoS One ; 10(5): e0126846, 2015.
Article in English | MEDLINE | ID: mdl-25962111

ABSTRACT

Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.


Subject(s)
Lung , Regeneration , Tissue Scaffolds , Alveolar Epithelial Cells/cytology , Animals , Apoptosis , Batch Cell Culture Techniques , Bioreactors , Cell Differentiation , Cell Proliferation , Female , Lung/physiology , Male , Mesenchymal Stem Cells/cytology , Mice , Models, Animal
5.
Emerg Infect Dis ; 19(3): 431-8, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23622517

ABSTRACT

Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide. An in vitro model for NoV replication remains elusive, making study of the virus difficult. A previous study, which used a 3-dimensional (3-D) intestinal model derived from INT-407 cells reported NoV replication and extensive cytopathic effects (CPE). Using the same 3-D model, but with highly purified Norwalk virus (NV), we attempted to replicate this study. Our results showed no evidence of NV replication by real-time PCR of viral RNA or by immunocytochemical detection of viral structural and nonstructural proteins. Immunocytochemical analysis of the 3-D cultures also showed no detectable presence of histo-blood group antigens that participate in NV binding and host tropism. To determine the potential cause of CPE observed in the previous study, we exposed 3-D cultures to lipopolysaccharide concentrations consistent with contaminated stool samples and observed morphologic features similar to CPE. We conclude that the 3-D INT-407 model does not support NV replication.


Subject(s)
Epithelial Cells/virology , Gastroenteritis/virology , Intestinal Mucosa/virology , Norovirus/physiology , Virus Replication , Blood Group Antigens/metabolism , Cell Aggregation , Cell Culture Techniques , Cell Line , Epithelial Cells/immunology , Gastroenteritis/immunology , Gastroenteritis/pathology , Humans , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Lipopolysaccharides/pharmacology , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Viral Nonstructural Proteins/metabolism , Viral Structural Proteins/metabolism , Viral Tropism
6.
PLoS One ; 7(5): e37116, 2012.
Article in English | MEDLINE | ID: mdl-22693569

ABSTRACT

The probiotic effects of Lactobacillus reuteri have been speculated to partly depend on its capacity to produce the antimicrobial substance reuterin during the reduction of glycerol in the gut. In this study, the potential of this process to protect human intestinal epithelial cells against infection with Salmonella enterica serovar Typhimurium was investigated. We used a three-dimensional (3-D) organotypic model of human colonic epithelium that was previously validated and applied to study interactions between S. Typhimurium and the intestinal epithelium that lead to enteric salmonellosis. Using this model system, we show that L. reuteri protects the intestinal cells against the early stages of Salmonella infection and that this effect is significantly increased when L. reuteri is stimulated to produce reuterin from glycerol. More specifically, the reuterin-containing ferment of L. reuteri caused a reduction in Salmonella adherence and invasion (1 log unit), and intracellular survival (2 log units). In contrast, the L. reuteri ferment without reuterin stimulated growth of the intracellular Salmonella population with 1 log unit. The short-term exposure to reuterin or the reuterin-containing ferment had no observed negative impact on intestinal epithelial cell health. However, long-term exposure (24 h) induced a complete loss of cell-cell contact within the epithelial aggregates and compromised cell viability. Collectively, these results shed light on a potential role for reuterin in inhibiting Salmonella-induced intestinal infections and may support the combined application of glycerol and L. reuteri. While future in vitro and in vivo studies of reuterin on intestinal health should fine-tune our understanding of the mechanistic effects, in particular in the presence of a complex gut microbiota, this the first report of a reuterin effect on the enteric infection process in any mammalian cell type.


Subject(s)
Colon/cytology , Dietary Supplements , Glycerol/pharmacology , Intestinal Mucosa/drug effects , Intestinal Mucosa/microbiology , Limosilactobacillus reuteri/physiology , Salmonella typhimurium/growth & development , Cell Survival/drug effects , Culture Media, Conditioned/metabolism , Fermentation/drug effects , Glyceraldehyde/analogs & derivatives , Glyceraldehyde/metabolism , Glycerol/metabolism , HT29 Cells , Humans , Intestinal Mucosa/cytology , Limosilactobacillus reuteri/drug effects , Limosilactobacillus reuteri/metabolism , Models, Molecular , Propane/metabolism , Salmonella typhimurium/physiology
7.
PLoS One ; 7(1): e29481, 2012.
Article in English | MEDLINE | ID: mdl-22238616

ABSTRACT

Extra-intestinal pathogenic E. coli (ExPEC), including avian pathogenic E. coli (APEC), pose a considerable threat to both human and animal health, with illness causing substantial economic loss. APEC strain χ7122 (O78∶K80∶H9), containing three large plasmids [pChi7122-1 (IncFIB/FIIA-FIC), pChi7122-2 (IncFII), and pChi7122-3 (IncI(2))]; and a small plasmid pChi7122-4 (ColE2-like), has been used for many years as a model strain to study the molecular mechanisms of ExPEC pathogenicity and zoonotic potential. We previously sequenced and characterized the plasmid pChi7122-1 and determined its importance in systemic APEC infection; however the roles of the other pChi7122 plasmids were still ambiguous. Herein we present the sequence of the remaining pChi7122 plasmids, confirming that pChi7122-2 and pChi7122-3 encode an ABC iron transport system (eitABCD) and a putative type IV fimbriae respectively, whereas pChi7122-4 is a cryptic plasmid. New features were also identified, including a gene cluster on pChi7122-2 that is not present in other E. coli strains but is found in Salmonella serovars and is predicted to encode the sugars catabolic pathways. In vitro evaluation of the APEC χ7122 derivative strains with the three large plasmids, either individually or in combinations, provided new insights into the role of plasmids in biofilm formation, bile and acid tolerance, and the interaction of E. coli strains with 3-D cultures of intestinal epithelial cells. In this study, we show that the nature and combinations of plasmids, as well as the background of the host strains, have an effect on these phenomena. Our data reveal new insights into the role of extra-chromosomal sequences in fitness and diversity of ExPEC in their phenotypes.


Subject(s)
Birds/microbiology , Escherichia coli/genetics , Genetic Fitness/genetics , Plasmids/genetics , Animals , Antigenic Variation/genetics , Base Sequence , Birds/genetics , Chickens/genetics , Chickens/microbiology , DNA, Bacterial/genetics , Escherichia coli/pathogenicity , Escherichia coli Infections/genetics , Escherichia coli Infections/veterinary , Immune Evasion/genetics , Models, Biological , Models, Molecular , Molecular Sequence Data , Plasmids/analysis , Poultry Diseases/genetics , Poultry Diseases/microbiology , Virulence Factors/chemistry , Virulence Factors/genetics
8.
Cell Microbiol ; 13(3): 469-81, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21054742

ABSTRACT

The quorum sensing signal N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C(12) HSL), produced by Pseudomonas aeruginosa, exerts cytotoxic effects in macrophages in vitro, which is believed to affect host innate immunity in vivo. However, the medical significance of this finding to pulmonary disease remains unclear since the multicellular complexity of the lung was not considered in the assessment of macrophage responses to 3-oxo-C(12) HSL. We developed a novel three-dimensional co-culture model of alveolar epithelium and macrophages using the rotating wall vessel (RWV) bioreactor, by adding undifferentiated monocytes to RWV-derived alveolar epithelium. Our three-dimensional model expressed important architectural/phenotypic hallmarks of the parental tissue, as evidenced by highly differentiated epithelium, spontaneous differentiation of monocytes to functional macrophage-like cells, localization of these cells on the alveolar surface and a macrophage-to-epithelial cell ratio relevant to the in vivo situation. Co-cultivation of macrophages with alveolar epithelium counteracted 3-oxo-C(12) HSL-induced cytotoxicity via removal of quorum sensing molecules by alveolar cells. Furthermore, 3-oxo-C(12) HSL induced the intercellular adhesion molecule ICAM-1 in both alveolar epithelium and macrophages. These data stress the importance of multicellular organotypic models to integrate the role of different cell types in overall lung homeostasis and disease development in response to external factors.


Subject(s)
4-Butyrolactone/analogs & derivatives , Homoserine/analogs & derivatives , Macrophages/physiology , Pulmonary Alveoli/immunology , Pulmonary Alveoli/physiology , Quorum Sensing , 4-Butyrolactone/metabolism , 4-Butyrolactone/toxicity , Coculture Techniques , Epithelial Cells/immunology , Epithelial Cells/physiology , Flow Cytometry , Homoserine/metabolism , Homoserine/toxicity , Humans , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Lung/immunology , Microscopy, Confocal , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/pathogenicity , Respiratory Mucosa/immunology , Signal Transduction , U937 Cells
9.
Nat Rev Microbiol ; 8(11): 791-801, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20948552

ABSTRACT

Appropriately simulating the three-dimensional (3D) environment in which tissues normally develop and function is crucial for engineering in vitro models that can be used for the meaningful dissection of host-pathogen interactions. This Review highlights how the rotating wall vessel bioreactor has been used to establish 3D hierarchical models that range in complexity from a single cell type to multicellular co-culture models that recapitulate the 3D architecture of tissues observed in vivo. The application of these models to the study of infectious diseases is discussed.


Subject(s)
Bioreactors , Host-Pathogen Interactions/physiology , Models, Biological , Animals , Biomedical Engineering , Cells, Cultured , Communicable Diseases/etiology , Humans , In Vitro Techniques , Rotation
10.
Biomaterials ; 31(32): 8426-35, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20692703

ABSTRACT

With the increasing necessity for functional tissue- and organ equivalents in the clinic, the optimization of techniques for the in vitro generation of organotypic structures that closely resemble the native tissue is of paramount importance. The engineering of a variety of highly differentiated tissues has been achieved using the rotating wall vessel (RWV) bioreactor technology, which is an optimized suspension culture allowing cells to grow in three-dimensions (3-D). However, certain cell types require the use of scaffolds, such as collagen-coated microcarrier beads, for optimal growth and differentiation in the RWV. Removal of the 3-D structures from the microcarriers involves enzymatic treatment, which disrupts the delicate 3-D architecture and makes it inapplicable for potential implantation. Therefore, we designed a microcarrier bead coated with a synthetic extracellular matrix (ECM) composed of a disulfide-crosslinked hyaluronan and gelatin hydrogel for 3-D tissue engineering, that allows for enzyme-free cell detachment under mild reductive conditions (i.e. by a thiol-disulfide exchange reaction). The ECM-coated beads (ECB) served as scaffold to culture human intestinal epithelial cells (Int-407) in the RWV, which formed viable multi-layered cell aggregates and expressed epithelial differentiation markers. The cell aggregates remained viable following dissociation from the microcarriers, and could be returned to the RWV bioreactor for further culturing into bead-free tissue assemblies. The developed ECBs thus offer the potential to generate scaffold-free 3-D tissue assemblies, which could further be explored for tissue replacement and remodeling.


Subject(s)
Bioreactors , Hyaluronic Acid/chemistry , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Cell Line , Chromosomes , Disulfides/chemistry , Epithelial Cells/cytology , Epithelial Cells/metabolism , Humans , Sulfhydryl Compounds/chemistry
11.
PLoS One ; 5(12): e15750, 2010 Dec 29.
Article in English | MEDLINE | ID: mdl-21206750

ABSTRACT

The prevailing paradigm of Salmonella enteropathogenesis based on monolayers asserts that Salmonella pathogenicity island-1 Type Three Secretion System (SPI-1 T3SS) is required for bacterial invasion into intestinal epithelium. However, little is known about the role of SPI-1 in mediating gastrointestinal disease in humans. Recently, SPI-1 deficient nontyphoidal Salmonella strains were isolated from infected humans and animals, indicating that SPI-1 is not required to cause enteropathogenesis and demonstrating the need for more in vivo-like models. Here, we utilized a previously characterized 3-D organotypic model of human intestinal epithelium to elucidate the role of all characterized Salmonella enterica T3SSs. Similar to in vivo reports, the Salmonella SPI-1 T3SS was not required to invade 3-D intestinal cells. Additionally, Salmonella strains carrying single (SPI-1 or SPI-2), double (SPI-1/2) and complete T3SS knockout (SPI-1/SPI-2: flhDC) also invaded 3-D intestinal cells to wildtype levels. Invasion of wildtype and TTSS mutants was a Salmonella active process, whereas non-invasive bacterial strains, bacterial size beads, and heat-killed Salmonella did not invade 3-D cells. Wildtype and T3SS mutants did not preferentially target different cell types identified within the 3-D intestinal aggregates, including M-cells/M-like cells, enterocytes, or Paneth cells. Moreover, each T3SS was necessary for substantial intracellular bacterial replication within 3-D cells. Collectively, these results indicate that T3SSs are dispensable for Salmonella invasion into highly differentiated 3-D models of human intestinal epithelial cells, but are required for intracellular bacterial growth, paralleling in vivo infection observations and demonstrating the utility of these models in predicting in vivo-like pathogenic mechanisms.


Subject(s)
Intestinal Mucosa/metabolism , Mutation , Salmonella enterica/genetics , Animals , Cell Line , Cells, Cultured , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Humans , Imaging, Three-Dimensional , Intestinal Mucosa/cytology , Mice , Mice, Knockout , Microscopy, Confocal/methods , Salmonella Infections/microbiology
12.
PLoS One ; 3(12): e3923, 2008.
Article in English | MEDLINE | ID: mdl-19079590

ABSTRACT

The spaceflight environment is relevant to conditions encountered by pathogens during the course of infection and induces novel changes in microbial pathogenesis not observed using conventional methods. It is unclear how microbial cells sense spaceflight-associated changes to their growth environment and orchestrate corresponding changes in molecular and physiological phenotypes relevant to the infection process. Here we report that spaceflight-induced increases in Salmonella virulence are regulated by media ion composition, and that phosphate ion is sufficient to alter related pathogenesis responses in a spaceflight analogue model. Using whole genome microarray and proteomic analyses from two independent Space Shuttle missions, we identified evolutionarily conserved molecular pathways in Salmonella that respond to spaceflight under all media compositions tested. Identification of conserved regulatory paradigms opens new avenues to control microbial responses during the infection process and holds promise to provide an improved understanding of human health and disease on Earth.


Subject(s)
Culture Media/chemistry , Gene Expression Regulation, Bacterial , Salmonella/genetics , Salmonella/pathogenicity , Space Flight , Animals , Genes, Bacterial , Ions , Lethal Dose 50 , Mice , Phosphates/metabolism , Proteomics , Reverse Transcriptase Polymerase Chain Reaction , Salmonella/growth & development , Transcription, Genetic
13.
Virology ; 335(1): 72-86, 2005 Apr 25.
Article in English | MEDLINE | ID: mdl-15823607

ABSTRACT

To determine if the cellular factors La autoantigen (La) and polypyrimidine tract-binding protein (PTB) are required for hepatitis C virus (HCV) replication, we used siRNAs to silence these factors and then monitored their effect on HCV replication using quantitative RT-PCR. In addition, we determined the influence of PTB on the activity of the 3' noncoding region (NCR) of HCV and investigated its interaction with the components of the HCV replicase complex. We found that La is essential for efficient HCV replication while PTB appears to partially repress replication. PTB does, however, block the binding of HCV RNA-dependent RNA polymerase (RdRp, NS5B) to the 3'NCR. Indirect immunofluorescence microscopy showed co-localization of cytoplasmic PTB with the HCV RdRp in hepatoma cells (Huh-7) expressing HCV proteins, while in vitro translation of viral proteins from the HCV replicon revealed the interaction of PTB isoforms with NS5B polymerase and NS3.


Subject(s)
Hepacivirus/physiology , Polypyrimidine Tract-Binding Protein/metabolism , Ribonucleoproteins/metabolism , Virus Replication , 3' Untranslated Regions/metabolism , Autoantigens , Base Sequence , Cell Line, Tumor , Humans , Molecular Sequence Data , Polypyrimidine Tract-Binding Protein/genetics , Protein Biosynthesis , RNA, Small Interfering/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleoproteins/genetics , Viral Nonstructural Proteins/metabolism , SS-B Antigen
14.
RNA ; 10(2): 321-9, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14730030

ABSTRACT

Positive-strand RNA viruses replicate their RNA genome within a ribonucleoprotein (RNP) complex that is associated with cellular membranes. We used a two-step method of purification to isolate hepatitis C virus (HCV) RNP complexes from human hepatoma cell line Huh7, which stably expresses HCV subgenomic replicons. The procedure involved hybridization of replicon-expressing cellular lysates with oligonucleotides tagged with biotin and digoxigenin at their respective termini complementary to subgenomic replicon RNA followed by avidin-agarose enrichment of the mixture and subsequent immunoprecipitation of biotin-eluted material with anti-digoxigenin antibody. The immunoprecipitates were immunoblotted with antisera against HCV nonstructural (NS) proteins. The analysis revealed the association of all the HCV NS proteins (NS3, NS4a, NS4b, NS5a, and NS5b) that are encoded by the subgenomic replicon RNA. The HCV RNP complex migrated in a native polyacrylamide gel with an approximate molecular mass of 450 kD. The association of these viral proteins in the RNP complex reinforces the widely acknowledged notion that RNA viruses accomplish replication within a membranous RNP complex.


Subject(s)
Chromatography, Affinity/methods , Hepacivirus/chemistry , Ribonucleoproteins/isolation & purification , Electrophoresis, Polyacrylamide Gel/methods , Humans , Precipitin Tests/methods , Tumor Cells, Cultured
15.
J Bacteriol ; 184(9): 2360-9, 2002 May.
Article in English | MEDLINE | ID: mdl-11948148

ABSTRACT

SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins. secA is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions. A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of secA, secM. The secM translational pause prevents formation of an RNA helix that normally blocks secA translational initiation. The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon. We characterized the atypical secM signal peptide and found that mutations within the amino-terminal region specifically affect the secM translational pause and secA regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and secA regulation. In addition, mutational analysis of the 3' end of secM allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level. Together, our results provide direct support for the secM translational pause model of secA regulation, and they pinpoint key sequences within secM that promote this important regulatory system.


Subject(s)
Adenosine Triphosphatases/genetics , Bacterial Proteins , Escherichia coli Proteins/genetics , Genes, Bacterial , Membrane Transport Proteins/genetics , Protein Biosynthesis , Protein Sorting Signals/genetics , 3' Untranslated Regions/genetics , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Enterobacteriaceae/genetics , Enterobacteriaceae/metabolism , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Membrane Transport Proteins/metabolism , Molecular Sequence Data , Mutation , Protein Sorting Signals/physiology , SEC Translocation Channels , SecA Proteins , Sequence Alignment
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