Deletion of a 19-Amino-Acid Region inClostridioides difficileTcdB2 Results in Spontaneous Autoprocessing and Reduced Cell Binding and Provides a Nontoxic Immunogen for Vaccination

ABSTRACTClostridioides difficiletoxin B (TcdB) is an intracellular toxin responsible for many of the pathologies ofC. difficileinfection. The two variant forms of TcdB (TcdB1 and TcdB2) share 92% sequence identity but have reported differences in rates of cell entry, autoprocessing, and overall toxicity. This 2,366-amino-acid, multidomain bacterial toxin glucosylates and inactivates small GTPases in the cytosol of target cells, ultimately leading to cell death. Successful cell entry and intoxication by TcdB are known to involve various conformational changes in the protein, including a proteolytic autoprocessing event. Previous studies found that amino acids 1753 to 1852 influence the conformational states of the proximal carboxy-terminal domain of TcdB and could contribute to differences between TcdB1 and TcdB2. In the current study, a combination of approaches was used to identify sequences within the region from amino acids 1753 to 1852 that influence the conformational integrity and cytotoxicity of TcdB2. Four deletion mutants with reduced cytotoxicity were identified, while one mutant, TcdB2Δ1769–1787, exhibited no detectable cytotoxicity. TcdB2Δ1769–1787underwent spontaneous autoprocessing and was unable to interact with CHO-K1 or HeLa cells, suggesting a potential change in the conformation of the mutant protein. Despite the putative alteration in structural stability, vaccination with TcdB2Δ1769–1787induced a TcdB2-neutralizing antibody response and protected againstC. difficiledisease in a mouse model. These findings indicate that the 19-amino-acid region spanning residues 1769 to 1787 in TcdB2 is crucial to cytotoxicity and the structural regulation of autoprocessing and that TcdB2Δ1769–1787is a promising candidate for vaccination.

Download Full-text

Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry

mSphere ◽

10.1128/msphere.00268-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 4

Author(s):

Jonathan J. Hunt ◽

Jason L. Larabee ◽

Jimmy D. Ballard

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Clostridium Difficile ◽

Cell Entry ◽

Cell Binding ◽

Content Type ◽

Amino Acid Region ◽

Antibiotic Associated Diarrhea ◽

Insight Into ◽

Acid Region

ABSTRACT TcdB is a major virulence factor produced by Clostridium difficile, a leading cause of antibiotic-associated diarrhea. Hypervirulent strains of C. difficile encode a variant of TcdB (TcdB2) that is more toxic than toxin derived from historical strains (TcdB1). Though TcdB1 and TcdB2 exhibit 92% overall identity, a 99-amino-acid region previously associated with cell entry and spanning amino acids 1753 to 1851 has only 77% sequence identity. Results from the present study indicate that the substantial sequence variation in this region could contribute to the differences in cell entry between TcdB1 and TcdB2 and possibly explain TcdB2’s heightened toxicity. Finally, during the course of these studies, an unusual aspect of TcdB cell entry was discovered wherein cell binding appeared to depend on endocytosis. These findings provide insight into TcdB’s variant forms and their mechanisms of cell entry. Clostridium difficile TcdB2 enters cells with a higher efficiency than TcdB1 and exhibits an overall higher level of toxicity. However, the TcdB2-specific sequences that account for more efficient cell entry have not been reported. In this study, we examined the contribution of carboxy-terminal sequence differences to TcdB activity by comparing the binding, uptake, and endosomal localization of TcdB1 and TcdB2 or selected recombinant fragments of these proteins. Our findings suggest that sequence differences in the amino acid 1753 to 1851 region proximal to the combined repetitive oligopeptide domain (CROP) support enhanced uptake of TcdB2 and localization of toxin in acidified endosomes. In the absence of this region, the CROP domains of both forms of the toxin exhibited similar levels of cell interaction, while the addition of amino acids 1753 to 1851 greatly increased toxin binding by only TcdB2. Moreover, the amino acid 1753 to 2366 fragment of TcdB2, but not TcdB1, accumulated to detectable levels in acidified endosomes. Unexpectedly, we discovered an unusual relationship between endocytosis and the efficiency of cell binding for TcdB1 and TcdB2 wherein inhibition of endocytosis by a chemical inhibitor or incubation at a low temperature resulted in a dramatic reduction in cell binding. These findings provide information on sequence variations that may contribute to differences in TcdB1 and TcdB2 toxicity and reveal a heretofore unknown connection between endocytosis and cell binding for this toxin. IMPORTANCE TcdB is a major virulence factor produced by Clostridium difficile, a leading cause of antibiotic-associated diarrhea. Hypervirulent strains of C. difficile encode a variant of TcdB (TcdB2) that is more toxic than toxin derived from historical strains (TcdB1). Though TcdB1 and TcdB2 exhibit 92% overall identity, a 99-amino-acid region previously associated with cell entry and spanning amino acids 1753 to 1851 has only 77% sequence identity. Results from the present study indicate that the substantial sequence variation in this region could contribute to the differences in cell entry between TcdB1 and TcdB2 and possibly explain TcdB2’s heightened toxicity. Finally, during the course of these studies, an unusual aspect of TcdB cell entry was discovered wherein cell binding appeared to depend on endocytosis. These findings provide insight into TcdB’s variant forms and their mechanisms of cell entry.

Download Full-text

A Peptide Region from Factor Va Increases Factor Xa Efficiency for Cleavage of Prothrombin.

Blood ◽

10.1182/blood.v104.11.3977.3977 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3977-3977

Author(s):

Melissa A. Blum ◽

Daniel O. Beck ◽

Michael Kalafatis

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Heavy Chain ◽

Membrane Surface ◽

Factor Xa ◽

Amino Acid Region ◽

Factor Va ◽

Prothrombinase Activity ◽

Prothrombin Activation ◽

Acid Region

Abstract The procoagulant enzymatic complex, prothrombinase, which is required for normal hemostasis, is composed of the enzyme, factor Xa, the protein cofactor, factor Va, associated on a cell surface in the presence of divalent metal ions. Incorporation of factor Va into prothrombinase and its interaction with factor Xa increases the catalytic efficiency of the enzyme by five orders of magnitude as compared to factor Xa alone. While the importance of the contribution of factor Va to the activity of factor Xa for rapid thrombin formation by prothrombinase at the place of vascular injury has been long established, the consequence of the interaction of the cofactor with the members of prothrombinase and the molecular mechanism by which factor Va accelerates prothrombin activation remains an enigma. Prothrombin is activated following two cleavages (Arg271/Arg320). Depending on the order of peptide bond cleavage different intermediates are formed. Factor Xa alone cleaves prothrombin sequentially, first at Arg271 to produce fragment 1•2 and prethrombin-2, followed by cleavage at Arg320 to produce fragment 1•2 and thrombin. The prothrombinase complex catalyzes the activation of prothrombin following the opposite pathway (Arg320 followed by Arg271), resulting in a formation of an active intermediate (meizothrombin) and a 300,000-fold increase in the rate of the overall reaction compared with the rate of prothrombin activation observed with factor Xa alone. We have shown that amino acid region 307–348 of factor Va heavy chain is critical for cofactor activity. A peptide containing this amino acid sequence (42 amino acids, N42R) was found to interact with fluorescently labeled factor Xa and to inhibit prothrombinase activity. Our present data show that N42R can be cross-linked to the heavy chain of membrane-bound factor Xa in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have also demonstrated that amino acid region 323–331 from N42R (AP4′) contains a binding site for factor Xa of factor Va heavy chain. Our present data show that a peptide containing amino acid residues 317–326 (AP3) inhibited both prothrombinase activity and the high affinity interaction of factor Va with factor Xa on the membrane surface. Moreover, we have found using site directed mutagenesis and recombinant factor Va that amino acids at the NH2-terminal end of AP4′ (i.e. residues 323–325, Glu-Tyr-Phe) are responsible for the inhibitory effect of AP3 and AP4′ and are crucial for the interaction of factor Va with factor Xa. A tripeptide with this sequence inhibited prothrombinase activity in an assay using a fluorescent thrombin inhibitor. To identify the effect of these peptides on factor Xa’s ability to cleave and activate prothrombin, we studied prothrombin activation by gel electrophoresis. The data demonstrated that several peptides that inhibited both the factor Va-factor Xa interaction on the membrane surface and prothrombinase activity, had the ability to accelerate cleavage of prothrombin by factor Xa alone, in the absence of factor Va. Specifically, N42R and AP3 were found to increase the rate of prothrombin consumption by factor Xa by approximately four-fold when compared to factor Xa acting alone. Both peptides induced acceleration in prethrombin-2 formation suggesting an increased in the rate of cleavage of prothrombin at Arg271. These data suggest that the binding of factor Va to factor Xa through amino acid region 323–331 alone produces an effect on factor Xa that increases its potency for cleavage at Arg271.

Download Full-text

Amino Acid Region 1000–1008 of Coagulation Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

Blood ◽

10.1182/blood.v116.21.2221.2221 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2221-2221

Author(s):

Joesph R Wiencek ◽

Jamila Hirbawi ◽

Mahesheema Na ◽

Michael Kalafatis

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Coagulation Factor ◽

Procoagulant Activity ◽

Factor V ◽

Prothrombinase Complex ◽

Amino Acid Region ◽

Coagulation Factor V ◽

Acid Region

Abstract Abstract 2221 The intricate process of hemostasis is a highly regulated mechanism which implements the conversion of prothrombin to thrombin and the crucial formation of a fibrin clot. The direct progression of hemostasis is pivotal to the prevention of various clotting disorders associated to hypercoagulation and excess bleeding. Upon vascular injury, the proteolytic conversion of prothrombin to thrombin compatible to rates of survival is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a phospholipid membrane in the presence of divalent metal ions. Coagulation factor V (fV) is synthesized as a multi-domain (A1-A2-B-A3-C1-C2) quiescent procofactor with nominal procoagulant activity. Following the three sequential catalytic cleavages by a-thrombin at Arg709, Arg1018 and Arg1545 amino acids 710–1545 of the B-domain are liberated to generate the noncovalently associated light and heavy chains of fVa. The cleavage at Arg1545 is crucial for full procoagulant activity. The heterodimer of fVa is composed of a heavy chain associated with the 2 A domains (residues 1–303 and 317–656) and a light chain composed of one A domain (1546-1877) and two C domains (residues 1878–2036 and 2037–2196). Since single chain fV does not bind fXa, the proper removal of the B-domain is vital to generate procoagulant activity. The incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. Appropriate binding of fVa to fXa during prothrombinase function is essential to the proper activation of the substrate, prothrombin. Previous studies have determined the heavy and light chains of fVa to have fXa interactive sites. A highly basic region of amino acids in the B-domain suggests a potential sheathing of either the heavy or light chain fXa interface sites. To verify this hypothesis we investigated the role of amino acid region 1000–1008 that contains seven basic amino acid residues. To ascertain the role of this region we have constructed a recombinant mutant fV molecule with all activation cleavage sites (R709/R1018/R1545) mutated to glutamine (fV*T3Q), a mutant fV molecule with region 1000–1008 deleted (fVΔ1000-1008), and a mutant fV molecule containing the same deletion with all activation cleavage sites changed to glutamine (fVΔ1000-1008/*T3Q). The recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells, purified to homogeneity, and assessed for their capability to bind fXa within prothrombinase prior (fV) and after incubation with thrombin (fVa). The data showed that fV*T3Q and fVa*T3Q were unable to interact with fXa. In contrast, the Kd values for fVΔ1000-1008 (0.9 nM), fVaΔ1000-1008 (0.4 nM), fVΔ1000-1008*T3Q (0.7 nM) and fVaΔ1000-1008*T3Q (0.5 nM), were similar to the affinity of fVaWT for fXa (0.22 nM). Two-stage clotting assays revealed that while fVa*T3Q was practically devoid of clotting activity, the mutant molecules fVaΔ1000-1008, and fVaD1000-1008*T3Q had clotting activities comparable to fVaWT. Thus, unactivated fVΔ1000-1008*T3Q has an affinity for fXa that is similar to the affinity of fVaWT for the enzyme. In addition, fVΔ1000-1008*T3Q that cannot be cleaved and activated by thrombin or activated during the course of the clotting assay, has similar clotting activity as fVaWT (∼3110 U/mg). The data presented in this study provide an important insight into one of the possible roles of the B domain of factor V, explicitly the fXa interactive sites on fVa are covered/inhibited by amino acids 1000–1008 of the fV B-domain. These data strongly suggest that amino acid region 1000–1008 of fV contains a regulatory sequence protecting the organisms from spontaneous binding of the procofactor to fXa and unnecessary prothrombinase complex formation which will result in catastrophic physiological consequences. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Mutational analysis defines a C-terminal tail domain of RAP1 essential for Telomeric silencing in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/138.4.1025 ◽

1994 ◽

Vol 138 (4) ◽

pp. 1025-1040 ◽

Cited By ~ 3

Author(s):

C Liu ◽

X Mao ◽

A J Lustig

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Size Control ◽

Telomere Maintenance ◽

Site Directed Mutagenesis ◽

In Vitro Mutagenesis ◽

Tail Domain ◽

Amino Acid Region ◽

Telomeric Silencing ◽

Acid Region

Abstract Alleles specifically defective in telomeric silencing were generated by in vitro mutagenesis of the yeast RAP1 gene. The most severe phenotypes occur with three mutations in the C-terminal 28 amino acids. Two of the alleles are nonsense mutations resulting in truncated repressor/activator protein 1 (RAP1) species lacking the C-terminal 25-28 amino acids; the third allele is a missense mutation within this region. These alleles define a novel 28-amino acid region, termed the C-terminal tail domain, that is essential for telomeric and HML silencing. Using site-directed mutagenesis, an 8-amino acid region (amino acids 818-825) that is essential for telomeric silencing has been localized within this domain. Further characterization of these alleles has indicated that the C-terminal tail domain also plays a role in telomere size control. The function of the C-terminal tail in telomere maintenance is not mediated through the RAP1 interacting factor RIF1: rap1 alleles defective in both the C-terminal tail and RIF1 interaction domains have additive effects on telomere length. Overproduction of SIR3, a dose-dependent enhancer of telomeric silencing, suppresses the telomeric silencing, but not length, phenotypes of a subset of C-terminal tail alleles. In contrast, an allele that truncates the terminal 28 amino acids of RAP1 is refractory to SIR3 overproduction. These results indicate that the C-terminal tail domain is required for SIR3-dependent enhancement of telomeric silencing. These data also suggest a distinct set of C-terminal requirements for telomere size control and telomeric silencing.

Download Full-text

Role of the N-Terminal Amino Acid Region of Factor Va Light Chain In Prothrombinase Assembly and Function.

Blood ◽

10.1182/blood.v116.21.1127.1127 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1127-1127

Author(s):

John L. Vaughn ◽

Jamila Hirbawi ◽

Michael A. Bukys ◽

Michael Kalafatis

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Light Chain ◽

Gel Electrophoresis ◽

Fold Increase ◽

Divalent Ions ◽

Amino Acid Region ◽

Factor Va ◽

And Function ◽

Acid Region

Abstract Abstract 1127 The timely activation of prothrombin (II) to thrombin (IIa) by the prothrombinase (IIase) complex is required for the maintenance of hemostasis. The IIase complex is composed of the enzyme factor Xa (fXa) and the cofactor factor Va (fVa) assembled on a membrane surface in the presence of divalent ions. While fXa alone is capable of activating II, the incorporation of fVa into the IIase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for IIa formation. FVa is generated following limited proteolysis of factor V (fV) by IIa at Arg709, Arg1018, and Arg1545, releasing a heavy chain (residues 1–709) and light chain (residues 1546–2196) that associate noncovalently through divalent ions. The A3 domain of the light chain of fVa has been implicated in the interaction of the cofactor with fXa. To determine the contribution of the A3 domain of fVa to IIase assembly and function, synthetic peptides representing residues 1546–1612 were screened for their ability to inhibit IIase. The peptide representing residues 1546–1558 strongly inhibited IIase with an IC50 of 50 μM. Additional fluorometric studies with overlapping pentapeptides from that region revealed that amino acids 1553–1558 were responsible for the observed inhibition. To verify the data from the overlapping pentapeptide studies, site-directed mutagenesis was used to generate a fVa mutant molecule with amino acids 1549–1558 deleted (fVaLCD1). FVaLCD1 demonstrated greatly reduced clotting activity in two-stage clotting assays. Gel electrophoresis of the IIase catalyzed activation of II using fVaLCD1 revealed a 50% reduction in the rate of prothrombin consumption. IIase assembled with fVaLCD1 exhibited a 5-fold increase in the apparent disassociation constant (Kdapp) for the enzyme-cofactor interaction and a 50% reduction in the turnover number (kcat) for the enzyme. To investigate the possibility that amino acids 1549–1558 are important for procofactor activation, the fVaLCD1 mutant was incubated with IIa and visualized with gel electrophoresis. The fVaLCD1 mutant exhibited a greatly reduced rate of IIa catalyzed activation. Additional recombinant fVa molecules with overlapping alanine mutations within the amino acid region 1548–1559 revealed that amino acids 1554–1555 were responsible for the observed effects in fVaLCD1. The recombinant fVa molecule with amino acids 1554–1555 mutated to alanines (fVaAA) demonstrated a greatly reduced clotting activity, an 8-fold increase in the Kdapp for the enzyme-cofactor interaction, and a 50% reduction in the kcat for the enzyme. To further investigate the mutational robustness of fVa against mutations in amino acids 1554–1555, a recombinant fVa molecule with both tyrosine residues in that region mutated conservatively to phenylalanines (fVaFF) was created. The more conservative fVaFF mutant, which differed from fVaWT only in the absence of side-chain hydroxyl moieties, exhibited normal clotting activity, kinetics, and procofactor activation. Altogether, the data demonstrate that amino acids 1554–1555 from the N-terminal region of the fVa light chain are important for IIase assembly and function because they constitute an interactive site for IIa and fXa. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Xrs2p Regulates Mre11p Translocation to the Nucleus and Plays a Role in Telomere Elongation and Meiotic Recombination

Molecular Biology of the Cell ◽

10.1091/mbc.e04-09-0782 ◽

2005 ◽

Vol 16 (2) ◽

pp. 597-608 ◽

Cited By ~ 44

Author(s):

Yasumasa Tsukamoto ◽

Chikako Mitsuoka ◽

Masahiro Terasawa ◽

Hideyuki Ogawa ◽

Tomoko Ogawa

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Binding Site ◽

Meiotic Recombination ◽

Cell Cycle Checkpoint ◽

Checkpoint Control ◽

Telomere Elongation ◽

Amino Acid Region ◽

C Terminus ◽

Acid Region

The Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in meiotic recombination, repair of damaged DNA, telomere elongation, and cell cycle checkpoint control. Xrs2p is known to be essential for all the functions of the complex, but its role in the complex has not been clearly elucidated. A 32-amino acid region near the C terminus of Xrs2p was identified as an Mre11p-binding site. No more function of Xrs2p than translocation of Mre11p from the cytoplasm to the nucleus is necessary for response to DNA damage. However, domains in Xrs2p located both 49 amino acids upstream and 104 amino acids downstream of the Mre11p binding site are required for meiotic recombination and telomere elongation, respectively, in addition to the 32-amino acid region. These findings demonstrate that Xrs2p acts as a specificity factor that allows the MRX complex to function in meiotic recombination and in telomere elongation.

Download Full-text

Defining the Functionally Important Domain and Amino Acid Residues in Mycobacterium tuberculosis Integration Host Factor for Genome Stability, DNA Binding, and Integrative Recombination

Journal of Bacteriology ◽

10.1128/jb.00357-17 ◽

2017 ◽

Vol 199 (19) ◽

Cited By ~ 2

Author(s):

Narayanaswamy Sharadamma ◽

Yadumurthy Harshavardhana ◽

K. Muniyappa

Keyword(s):

Dna Damage ◽

Mycobacterium Tuberculosis ◽

Amino Acid ◽

Dna Binding ◽

Integration Host Factor ◽

Host Factor ◽

Content Type ◽

Amino Acid Region ◽

N Terminus ◽

Acid Region

ABSTRACT The integration host factor of Mycobacterium tuberculosis (mIHF) consists of a single polypeptide chain, the product of the ihf gene. We previously revealed that mIHF is a novel member of a new class of nucleoid-associated proteins that have important roles in DNA damage response, nucleoid compaction, and integrative recombination. The mIHF contains a region of 86 amino acids at its N terminus, absent from both α- and β-subunits of Escherichia coli IHF. However, the functional significance of an extra 86-amino-acid region in the full-length protein remains unknown. Here, we report the structure/function relationship of the DNA-binding and integrative recombination-stimulating activity of mIHF. Deletion mutagenesis showed that an extra 86-amino-acid region at the N terminus is dispensable; the C-terminal region possesses the sequences essential for its known biological functions, including the ability to suppress the sensitivity of E. coli ΔihfA and ΔihfB cells to DNA-damaging agents, DNA binding, DNA multimerization-circularization, and stimulation of phage L5 integrase-catalyzed integrative recombination. Single and double alanine substitutions at positions Arg170 and Arg171, located at the mIHF DNA-binding site, abrogated its capacity to suppress the sensitivity of E. coli ΔihfA and ΔihfB cells to DNA-damaging agents. The variants encoded by these mutant alleles failed to bind DNA and stimulate integrative recombination. Interestingly, the DNA-binding activity of the mIHF-R173A variant remained largely unaffected; however, it was unable to stimulate integrative recombination, thus revealing a separation-of-function allele of mIHF. The functional and structural characterization of this separation-of-function allele of mIHF could reveal previously unknown functions of IHF. IMPORTANCE The integration host factor of Mycobacterium tuberculosis is a novel nucleoid-associated protein. mIHF plays a vital role in DNA damage response, nucleoid compaction, and integrative recombination. Intriguingly, mIHF contains an extra 86-amino-acid region at its N terminus, absent from both α- and β-subunits of Escherichia coli IHF, whose functional significance is unknown. Furthermore, a triad of arginine residues located at the mIHF-DNA interface have been implicated in a range of its functions. Here, we reveal the roles of N- and C-terminal regions of mIHF and the individual residues in the Arg triad for their ability to provide protection in vivo against DNA damage, bind DNA, and stimulate integrase-catalyzed site-specific recombination.

Download Full-text

Identification of a GP64 Subdomain Involved in Receptor Binding by Budded Virions of the Baculovirus Autographica californica Multicapsid Nucleopolyhedrovirus

Journal of Virology ◽

10.1128/jvi.02490-07 ◽

2008 ◽

Vol 82 (9) ◽

pp. 4449-4460 ◽

Cited By ~ 52

Author(s):

Jian Zhou ◽

Gary W. Blissard

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Receptor Binding ◽

Envelope Glycoprotein ◽

Cell Receptor ◽

Single Amino Acid ◽

Virus Infectivity ◽

Amino Acid Region ◽

Host Cell Receptor ◽

Acid Region

ABSTRACT Enveloped virus entry into host cells is typically initiated by an interaction between a viral envelope glycoprotein and a host cell receptor. For budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, the envelope glycoprotein GP64 is involved in host cell receptor binding, and GP64 is sufficient to mediate low-pH-triggered membrane fusion. To better define the role of GP64 in receptor binding, we generated and characterized a panel of antisera against subdomains of GP64. Eight subdomain-specific antisera were generated, and their reactivities with GP64 proteins and neutralization of virus infectivity and binding were examined. Antibodies directed against the N-terminal region of GP64 (amino acids 21 to 159) showed strong neutralization of infectivity and effectively inhibited binding of 35S-labeled budded virions to Sf9 cells. In addition, we generated virions displaying truncated GP64 constructs. A construct displaying the N-terminal 274 amino acids (residues 21 to 294) of the ectodomain was sufficient to mediate virion binding. Additional studies of antisera directed against small subdomains revealed that an antiserum against a 40-amino-acid region (residues 121 to 160) neutralized virus infectivity. Site-directed mutagenesis was subsequently used for functional analysis of that region. Recombinant viruses expressing GP64 proteins with single amino acid substitutions within amino acids 120 to 124 and 142 to 148 replicated to high titers, suggesting that those amino acids were not critical for receptor binding or other important GP64 functions. In contrast, GP64 proteins with single amino acid substitutions of residues 153 and 156 were unable to substitute for wild-type GP64 and did not rescue a gp64 knockout virus. Further analysis showed that these substitutions substantially reduced binding of recombinant virus to Sf9 cells. Thus, the amino acid region from positions 21 to 159 was identified as a putative receptor binding domain, and amino acids 153 and 156 appear to be important for receptor binding.

Download Full-text

Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽

10.1128/mbio.00351-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 1

Author(s):

Rajdeep Banerjee ◽

Erin Weisenhorn ◽

Kevin J. Schwartz ◽

Kevin S. Myers ◽

Jeremy D. Glasner ◽

...

Keyword(s):

Amino Acids ◽

Urinary Tract ◽

Amino Acid ◽

Regulatory Network ◽

Iron Homeostasis ◽

Iron Limitation ◽

Content Type ◽

E Coli ◽

General Stress ◽

K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

Download Full-text

The Carboxy-Terminal Region ofFlavobacterium johnsoniaeSprB Facilitates Its Secretion by the Type IX Secretion System and Propulsion by the Gliding Motility Machinery

Journal of Bacteriology ◽

10.1128/jb.00218-19 ◽

2019 ◽

Vol 201 (19) ◽

Cited By ~ 5

Author(s):

Surashree S. Kulkarni ◽

Joseph J. Johnston ◽

Yongtao Zhu ◽

Zachary T. Hying ◽

Mark J. McBride

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cell Surface ◽

Type A ◽

Secretion System ◽

Gliding Motility ◽

Foreign Protein ◽

Type B ◽

Content Type ◽

Type Ix Secretion System

ABSTRACTFlavobacterium johnsoniaeSprB moves rapidly along the cell surface, resulting in gliding motility. SprB secretion requires the type IX secretion system (T9SS). Proteins secreted by the T9SS typically have conserved C-terminal domains (CTDs) belonging to the type A CTD or type B CTD family. Attachment of 70- to 100-amino-acid type A CTDs to a foreign protein allows its secretion. Type B CTDs are common but have received little attention. Secretion of the foreign protein superfolder green fluorescent protein (sfGFP) fused to regions spanning the SprB type B CTD (sfGFP-CTDSprB) was analyzed. CTDs of 218 amino acids or longer resulted in secretion of sfGFP, whereas a 149-amino-acid region did not. Some sfGFP was secreted in soluble form, whereas the rest was attached on the cell surface. Surface-attached sfGFP was rapidly propelled along the cell, suggesting productive interaction with the motility machinery. This did not result in rapid cell movement, which apparently requires additional regions of SprB. Secretion of sfGFP-CTDSprBrequired coexpression withsprF, which lies downstream ofsprB. SprF is similar in sequence toPorphyromonas gingivalisPorP. MostF. johnsoniaegenes encoding proteins with type B CTDs lie immediately upstream ofporP/sprF-like genes. sfGFP was fused to the type B CTD from one such protein (Fjoh_3952). This resulted in secretion of sfGFP only when it was coexpressed with its cognate PorP/SprF-like protein. These results highlight the need for extended regions of type B CTDs and for coexpression with the appropriate PorP/SprF-like protein for efficient secretion and cell surface localization of cargo proteins.IMPORTANCETheF. johnsoniaegliding motility adhesin SprB is delivered to the cell surface by the type IX secretion system (T9SS) and is rapidly propelled along the cell by the motility machinery. How this 6,497-amino-acid protein interacts with the secretion and motility machines is not known. Fusion of the C-terminal 218 amino acids of SprB to a foreign cargo protein resulted in its secretion, attachment to the cell surface, and rapid movement by the motility machinery. Efficient secretion of SprB required coexpression with the outer membrane protein SprF. Secreted proteins that have sequence similarity to SprB in their C-terminal regions are common in the phylumBacteroidetesand may have roles in adhesion, motility, and virulence.

Download Full-text