scholarly journals Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

2020 ◽  
Author(s):  
Christopher Icke ◽  
Freya J. Hodges ◽  
Karthik Puella ◽  
Samantha A. McKeand ◽  
Jack A. Bryant ◽  
...  
Keyword(s):  
Composite System ◽  
Cysteine Residue ◽  
Secretion System ◽  
Type I ◽  
Secretion Systems ◽  
Cellular Functions ◽  
E Coli ◽  
New Class ◽  
Secretion Pathway ◽  
Bacterial Lipoprotein

AbstractProtein acylation is critical for many cellular functions including signal transduction, cell division and development. In bacteria, such lipoproteins have important roles in virulence and are therefore potential targets for the development of novel antimicrobials and vaccines. To date, all known bacterial lipoproteins are secreted from the cytosol via the Sec pathway, acylated on an N-terminal cysteine residue through the action of Lgt, Lsp and Lnt, and then targeted to the appropriate cellular location. In the case of Gram-negative bacteria, the lipoprotein trafficking Lol pathway transports the lipoproteins to the outer membrane where most substrate molecules are retained within the cell. Here we identify a new secretion pathway that displays the substrate lipoprotein on the cell surface. We demonstrate that the previously identified E. coli Aat secretion system is a composite system that shares similarity with type I secretion systems and elements of the Lol pathway. Remarkably, during secretion by the Aat system, the AatD subunit acylates the substrate CexE on a highly conserved N-terminal glycine residue (rather than the canonical cysteine). Mutations in AatD or CexE that disrupt glycine acylation interfere with membrane incorporation and trafficking. Our data suggest that CexE is the first member of a new class of glycine-acylated bacterial lipoprotein, while Aat represents a new secretion system that we propose be defined as a lipoprotein secretion system (LSS).

scholarly journals Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Christopher Icke ◽  
Freya J Hodges ◽  
Karthik Pullela ◽  
Samantha A McKeand ◽  
Jack Alfred Bryant ◽  
...  
Keyword(s):  
Composite System ◽  
Cysteine Residue ◽  
Secretion System ◽  
Type I ◽  
Gram Negative Bacteria ◽  
Secretion Systems ◽  
Cellular Functions ◽  
New Class ◽  
Domains Of Life ◽  
Protein Acylation

Protein acylation is critical for many cellular functions across all domains of life. In bacteria, lipoproteins have important roles in virulence and are targets for the development of antimicrobials and vaccines. Bacterial lipoproteins are secreted from the cytosol via the Sec pathway and acylated on an N-terminal cysteine residue through the action of three enzymes. In Gram-negative bacteria, the Lol pathway transports lipoproteins to the outer membrane. Here we demonstrate that the Aat secretion system is a composite system sharing similarity with elements of a type I secretion systems and the Lol pathway. During secretion, the AatD subunit acylates the substrate CexE on a highly conserved N-terminal glycine residue. Mutations disrupting glycine acylation interfere with membrane incorporation and trafficking. Our data reveal CexE as the first member of a new class of glycine-acylated lipoprotein, while Aat represents a new secretion system that displays the substrate lipoprotein on the cell surface.

scholarly journals An A/U-Rich Enhancer Region Is Required for High-Level Protein Secretion through the HlyA Type I Secretion System

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Sakshi Khosa ◽  
Romy Scholz ◽  
Christian Schwarz ◽  
Mirko Trilling ◽  
Hartmut Hengel ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Protein Secretion ◽  
Fusion Proteins ◽  
Untranslated Region ◽  
Secretion System ◽  
Type I ◽  
Secretion Systems ◽  
Enhancer Region ◽  
Ribosomal Protein S1 ◽  
Cellular Expression

ABSTRACTEfficient protein secretion is often a valuable alternative to classic cellular expression to obtain homogenous protein samples. Early on, bacterial type I secretion systems (T1SS) were employed to allow heterologous secretion of fusion proteins. However, this approach was not fully exploited, as many proteins could not be secreted at all or only at low levels. Here, we present an engineered microbial secretion system which allows the effective production of proteins up to a molecular mass of 88 kDa. This system is based on the hemolysin A (HlyA) T1SS of the Gram-negative bacteriumEscherichia coli, which exports polypeptides when fused to a hemolysin secretion signal. We identified an A/U-rich enhancer region upstream ofhlyArequired for effective expression and secretion of selected heterologous proteins irrespective of their prokaryotic, viral, or eukaryotic origin. We further demonstrate that the ribosomal protein S1 binds to thehlyAA/U-rich enhancer region and that this region is involved in the high yields of secretion of functional proteins, like maltose-binding protein or human interferon alpha-2.IMPORTANCEA 5′ untranslated region of the mRNA of substrates of type I secretion systems (T1SS) drastically enhanced the secretion efficiency of the endogenously secreted protein. The identification of ribosomal protein S1 as the interaction partner of this 5′ untranslated region provides a rationale for the enhancement. This strategy furthermore can be transferred to fusion proteins allowing a broader, and eventually a more general, application of this system for secreting heterologous fusion proteins.

scholarly journals Escherichia coli type III secretion system 2 (ETT2) is widely distributed in avian pathogenic Escherichia coli isolates from Eastern China

2016 ◽  
Vol 144 (13) ◽  
pp. 2824-2830 ◽  
Author(s):  
S. WANG ◽  
X. LIU ◽  
X. XU ◽  
Y. ZHAO ◽  
D. YANG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Bacterial Infections ◽  
Eastern China ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Secretion Systems ◽  
E Coli ◽  
Type Iii ◽  
System 2

SUMMARYPathogens utilize type III secretion systems to deliver effector proteins, which facilitate bacterial infections. The Escherichia coli type III secretion system 2 (ETT2) which plays a crucial role in bacterial virulence, is present in the majority of E. coli strains, although ETT2 has undergone widespread mutational attrition. We investigated the distribution and characteristics of ETT2 in avian pathogenic E. coli (APEC) isolates and identified five different ETT2 isoforms, including intact ETT2, in 57·6% (141/245) of the isolates. The ETT2 locus was present in the predominant APEC serotypes O78, O2 and O1. All of the ETT2 loci in the serotype O78 isolates were degenerate, whereas an intact ETT2 locus was mostly present in O1 and O2 serotype strains, which belong to phylogenetic groups B2 and D, respectively. Interestingly, a putative second type III secretion-associated locus (eip locus) was present only in the isolates with an intact ETT2. Moreover, ETT2 was more widely distributed in APEC isolates and exhibited more isoforms compared to ETT2 in human extraintestinal pathogenic E. coli, suggesting that APEC might be a potential risk to human health. However, there was no distinct correlation between ETT2 and other virulence factors in APEC.

scholarly journals Genotype Cluster Analysis in PathogenicEscherichia coliIsolates Producing Different CDT Types

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Maryam Javadi ◽  
Mana Oloomi ◽  
Saeid Bouzari
Keyword(s):  
Virulence Genes ◽  
Type I ◽  
Cytolethal Distending Toxin ◽  
Chain Reaction ◽  
Shiga Toxins ◽  
E Coli ◽  
Secretion Pathway ◽  
Genotype Cluster ◽  
Catalase Peroxidase ◽  
Polymerase Chain

Diarrheagenic and uropathogenicE. colitypes are mainly characterized by the expression of distinctive bacterial virulent factors.stx1,stx2(Shiga toxins), andcdt(cytolethal distending toxin) genes have been acquired by horizontal gene transfer. Some virulent genes such asespP(serine protease),etpD(part of secretion pathway), andkatP(catalase-peroxidase), orsfpAgene (Sfp fimbriae), are on plasmids and the others likefliC(flagellin) and thefimHgene (fimbriae type-I) are located on chromosome. Genomic pathogenicity islands (PAIs) carry some virulent genes such ashlygene. To determine the existence of virulence genes incdtclinical isolates, genes includingstx1,stx2,cdt,hly,espP,katP,sfpA,etpD,fliC, andfimHwere assessed by Polymerase Chain Reaction (PCR). The most prevalent isolates foretpDandkatPgenes were 85.7% incdtII.katPgene was also observed 83.3% incdtI. However, in 42.85% ofcdtIIIisolates,espPgene was the most detected. Moreover,hlygene was also the most prominent gene incdtIII(71.42%).sfpA genewas observed in 66.6% ofcdtV.stx1gene was detected in 100% ofcdtII,cdtIV, andcdtVtypes. Presence and pattern of virulence genes were considered amongcdtpositive isotypes and used for their clustering and profiling.

scholarly journals Scale‐up of a Type I secretion system in E. coli using a defined mineral medium

2020 ◽  
Vol 36 (2) ◽  
Author(s):  
Nina Ihling ◽  
Andreas Uhde ◽  
Romy Scholz ◽  
Christian Schwarz ◽  
Lutz Schmitt ◽  
...  
Keyword(s):  
Scale Up ◽  
Secretion System ◽  
Mineral Medium ◽  
Type I ◽  
E Coli

scholarly journals Mechanism of Action of the Arylomycin Antibiotics and Effects of Signal Peptidase I Inhibition

2012 ◽  
Vol 56 (10) ◽  
pp. 5054-5060 ◽  
Author(s):  
Peter A. Smith ◽  
Floyd E. Romesberg
Keyword(s):  
Secretory Pathway ◽  
Signal Peptidase ◽  
Model Organisms ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Secretion Pathway ◽  
Secretion Stress ◽  
Signal Peptidase I ◽  
Essential Enzyme

ABSTRACTClinically approved antibiotics inhibit only a small number of conserved pathways that are essential for bacterial viability, and the physiological effects of inhibiting these pathways have been studied in great detail. Likewise, characterizing the effects of candidate antibiotics that function via novel mechanisms of action is critical for their development, which is of increasing importance due to the ever-growing problem of resistance. The arylomycins are a novel class of natural-product antibiotics that act via the inhibition of type I signal peptidase (SPase), which is an essential enzyme that functions as part of the general secretory pathway and is not the target of any clinically deployed antibiotic. Correspondingly, little is known about the effects of SPase inhibition or how bacteria may respond to mitigate the associated secretion stress. Using genetically sensitizedEscherichia coliandStaphylococcus aureusas model organisms, we examine the activity of arylomycin as a function of its concentration, bacterial cell density, target expression levels, and bacterial growth phase. The results reveal that the activity of the arylomycins results from an insufficient flux of proteins through the secretion pathway and the resulting mislocalization of proteins. Interestingly, this has profoundly different effects onE. coliandS. aureus. Finally, we examine the activity of arylomycin in combination with distinct classes of antibiotics and demonstrate that SPase inhibition results in synergistic sensitivity when combined with an aminoglycoside.

scholarly journals A highly conserved complete accessoryEscherichia colitype III secretion system 2 is widespread in bloodstream isolates of the ST69 lineage

2018 ◽  
Author(s):  
S. Fox ◽  
C. Goswami ◽  
M. Holden ◽  
J.P.R. Connolly ◽  
A. Roe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Secretion Systems ◽  
Functional Importance ◽  
Effective Prevention ◽  
E Coli ◽  
Type Iii ◽  
System 2

AbstractBacterial type III secretion systems (T3SS) play an important role in pathogenesis of Gram-negative infections. Enteropathogenic and enterohemorrhagicEscherichia colicontain a well-defined T3SS but in addition a second T3SS termedE. coliT3SS 2 (ETT2) has been described in a number of strains ofE. coli.The majority ofE. colicontain elements of a genetic locus encoding ETT2, but which has undergone significant mutational attrition rendering it without predicted function. Only a very few strains have been reported to contain an intact ETT2 locus. To investigate the occurrence of the ETT2 locus in strains of human pathogenicE. coli, we carried out genomic sequencing of 162 isolates obtained from patient blood cultures in Scotland. We found that all 26 ST69 isolates from this collection contained an intact ETT2 together with an associatedeiplocus which encodes putative secreted ETT2 effectors as well aseilA, a gene encoding a putative transcriptional regulator of ETT2 associated genes. Using a reporter gene foreilAactivation, we defined conditions under which this gene was differentially activated. However, comparison of secreted proteins from ST69 strains under high and loweilAactivation failed to identify any ETT2 secreted substrates. The conservation of the genes encoding ETT2 in human pathogenic ST69 strains strongly suggests it has functional importance in infection, although its exact functional role remains obscure.ImportanceOne of the commonest bacteria causing bloodstream infections in humans isEscherichia coli, which has a significant morbidity and mortality. Better understating of the mechanisms by which this microbe can invade blood could lead to more effective prevention and treatment. One mechanism by which some strains cause disease is by elaboration of a specialized secretion system, the type III secretion system (T3SS), encoded by the locus of enterocyte effacement (LEE). In addition to this well-defined T3SS, a second T3SS has been found in someE. colistrains termedE. colitype III secretion system 2 (ETT2). Most strains carry elements of the ETT2 locus, but with significant mutational attrition rendering it functionless. The significance of our work is that we have discovered that human bloodstream isolates ofE. coliof sequence type 69 contain a fully intact ETT2 and associated genes, strongly suggesting its functional importance in human infection.

scholarly journals The Caulobacter crescentus Paracrystalline S-Layer Protein Is Secreted by an ABC Transporter (Type I) Secretion Apparatus

1998 ◽  
Vol 180 (12) ◽  
pp. 3062-3069 ◽  
Author(s):  
Peter Awram ◽  
John Smit
Keyword(s):  
Abc Transporter ◽  
Transport System ◽  
Protein Transport ◽  
Caulobacter Crescentus ◽  
Sequence Similarity ◽  
Secretion System ◽  
Cell Protein ◽  
Type I ◽  
Secretion Systems ◽  
Secretion Signal

ABSTRACT Caulobacter crescentus is a gram-negative bacterium that produces a two-dimensional crystalline array on its surface composed of a single 98-kDa protein, RsaA. Secretion of RsaA to the cell surface relies on an uncleaved C-terminal secretion signal. In this report, we identify two genes encoding components of the RsaA secretion apparatus. These components are part of a type I secretion system involving an ABC transporter protein. These genes, lying immediately 3′ of rsaA, were found by screening a Tn5 transposon library for the loss of RsaA transport and characterizing the transposon-interrupted genes. The two proteins presumably encoded by these genes were found to have significant sequence similarity to ABC transporter and membrane fusion proteins of other type I secretion systems. The greatest sequence similarity was found to the alkaline protease (AprA) transport system ofPseudomonas aeruginosa and the metalloprotease (PrtB) transport system of Erwinia chrysanthemi. TheprtB and aprA genes were introduced intoC. crescentus, and their products were secreted by the RsaA transport system. Further, defects in the S-layer protein transport system led to the loss of this heterologous secretion. This is the first report of an S-layer protein secreted by a type I secretion apparatus. Unlike other type I secretion systems, the RsaA transport system secretes large amounts of its substrate protein (it is estimated that RsaA accounts for 10 to 12% of the total cell protein). Such levels are expected for bacterial S-layer proteins but are higher than for any other known type I secretion system.

scholarly journals Functional Type 1 Secretion System Involved in Legionella pneumophila Virulence

2014 ◽  
Vol 197 (3) ◽  
pp. 563-571 ◽  
Author(s):  
Fabien Fuche ◽  
Anne Vianney ◽  
Claire Andrea ◽  
Patricia Doublet ◽  
Christophe Gilbert
Keyword(s):  
Legionella Pneumophila ◽  
Severe Form ◽  
Secretion System ◽  
Host Cells ◽  
Type I ◽  
Secretion Systems ◽  
Content Type ◽  
Legionnaires Disease ◽  
Infectious Cycle

Legionella pneumophilais a Gram-negative pathogen found mainly in water, either in a free-living form or within infected protozoans, where it replicates. This bacterium can also infect humans by inhalation of contaminated aerosols, causing a severe form of pneumonia called legionellosis or Legionnaires' disease. The involvement of type II and IV secretion systems in the virulence ofL. pneumophilais now well documented. Despite bioinformatic studies showing that a type I secretion system (T1SS) could be present in this pathogen, the functionality of this system based on the LssB, LssD, and TolC proteins has never been established. Here, we report the demonstration of the functionality of the T1SS, as well as its role in the infectious cycle ofL. pneumophila. Using deletion mutants and fusion proteins, we demonstrated that therepeats-in-toxin protein RtxA is secreted through an LssB-LssD-TolC-dependent mechanism. Moreover, fluorescence monitoring and confocal microscopy showed that this T1SS is required for entry into the host cell, although it seems dispensable to the intracellular cycle. Together, these results underline the active participation ofL. pneumophila, via its T1SS, in its internalization into host cells.

scholarly journals Characterization of a Novel Microcin That Kills Enterohemorrhagic Escherichia coli O157:H7 and O26

2012 ◽  
Vol 78 (18) ◽  
pp. 6592-6599 ◽  
Author(s):  
Lauren J. Eberhart ◽  
James R. Deringer ◽  
Kelly A. Brayton ◽  
Ashish A. Sawant ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enterohemorrhagic Escherichia Coli ◽  
Logarithmic Phase ◽  
Type I ◽  
Escherichia Coli O157 ◽  
Content Type ◽  
E Coli ◽  
Secretion Pathway ◽  
Dependent Inhibition

ABSTRACTA novel phenotype was recently identified in which specific strains ofEscherichia coliinhibit competingE. colistrains via a mechanism that was designated “proximity-dependent inhibition” (PDI). PDI-expressing (PDI+)E. coliis known to inhibit susceptible (PDI−)E. colistrains, including several enterohemorrhagic (EHEC) and enterotoxigenic (ETEC)E. colistrains. In this study, every strain from a genetically diverse panel ofE. coliO157:H7 (n= 25) and additional strains ofE. coliserovar O26 were susceptible to the PDI phenotype. LIVE/DEAD staining was consistent with inhibition by killing of susceptible cells. Comparative genome analysis identified the genetic component of PDI, which is composed of a plasmid-borne (Incl1) operon encoding a putative microcin and associated genes for transport, immunity, and microcin activation. Transfer of the plasmid to a PDI−strain resulted in transfer of the phenotype, and deletion of the genes within the operon resulted in loss of the inhibition phenotype. Deletion of chromosomally encodedtolCalso resulted in loss of the inhibitory phenotype, and this confirmed that the putative microcin is most likely secreted via a type I secretion pathway. Deletion of an unrelated plasmid gene did not affect the PDI phenotype. Quantitative reverse transcription (RT)-PCR demonstrated that microcin expression is correlated with logarithmic-phase growth. The ability to inhibit a diversity ofE. colistrains indicates that this microcin may influence gut community composition and could be useful for control of important enteric pathogens.

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