Characterization of the hemolysin transporter, HlyB, using an epitope insertion.

Juranka, Peter F.; Zhang, F; Kulpa, Justyna; Endicott, J.A.; Blight, Mark A.; Holland, I. B.; Ling, Victor

doi:10.1016/s0021-9258(19)50591-9

Cited by 34 publications

(4 citation statements)

References 26 publications

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“…ABC transporters are composed of a highly conserved functional unit which includes two ATP binding domains and approximately 12 transmembrane segments. These transporters move a wide range of compounds across a variety of biological membranes (Croop, 1993;Juranka et al, 1992).…”

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confidence: 99%

Phosphatidylcholine and Phosphatidylethanolamine Behave as Substrates of the Human MDR1 P-Glycoprotein

et al. 1997

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The multidrug resistant cell line CEM/VBL300 and the parental CEM T-lymphoblastic cell line from which it was derived were used to study the accumulation of fluorescent phospholipid analogs of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). The fluorescent analogs NBD-PC, NBD-PE, and NBD-PS and [3H]PC were delivered in liposomes prepared by ethanol injection. Fluorescence microscopy demonstrated decreased accumulation of the NBD-PC analog in the multidrug resistant cell line compared to the parental cell line. Verapamil enhanced NBD-PC accumulation in the resistant cells. Similar results were obtained with insect cells expressing high levels of recombinant human MDR1. Elimination of NBD fluorescence on the outer leaflet of the plasma membrane with dithionite permitted quantification of the internal cellular fluorescence by FACS analysis. The drug resistant CEM/VBL300 cells accumulated approximately 10% the amount of NBD-PE and 20% the amount of NBD-PC compared to CEM drug sensitive cells. No difference in internal accumulation of NBD-PS was found between the drug resistant and drug sensitive cell lines. The internal accumulation of NBD-PE and NBD-PC was enhanced by the MDR reversal agents verapamil, cyclosporin A, and SDZ PSC 833 in the CEM/VBL300 cells but not in the CEM cells. The increased accumulation was dose dependent, and the relative potency of the reversal agents paralleled their ability to circumvent multidrug resistance. In addition, the monoclonal antibody UIC2 directed against the P-glycoprotein produced similar results. The evidence presented here suggests that PC and PE but not PS behave as substrates for human MDR1 P-glycoprotein.

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confidence: 99%

Phosphatidylcholine and Phosphatidylethanolamine Behave as Substrates of the Human MDR1 P-Glycoprotein

et al. 1997

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“…The signal for transport is located within the C-terminal 60 amino acids of HlyA (Koronakis et al, 1989;Hess et al, 1990;Stanley et al, 1991), which is not cleaved upon translocation (Felmlee et al, 1985). The transport is mediated by three proteins: two inner membrane proteins, HlyB and HlyD, encoded in the hemolysin operon, and an E. coli outer membrane protein TolC (Juranka et al, 1992;Wang et al, 1991;. There are several HlyB/D like transporters in gram negative bacteria responsible for the secretion of toxins such as Pasteurella leukotoxin, Bordetella cyclolysin, E. coli colicin V, and Erwinia metalloprotease.…”

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confidence: 99%

Secretion and Circular Dichroism Analysis of the C-Terminal Signal Peptides of HlyA and LktA

Zhang¹,

Yin²,

Arrowsmith³

et al. 1995

Biochemistry

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The secretion of the 107 kDa hemolysin A (HlyA) from Escherichia coli is mediated by membrane proteins hemolysin B (HlyB) and hemolysin D (HlyD). The signal for transport has been mapped to the C-terminal 60 amino acids of the HylA molecule. We have shown previously that the C-terminal 70 amino acids of leukotoxin (LktA) from Pasteurella hemolytica can substitute functionally for the HlyA signal sequence. This 70 amino acid peptide contains little primary sequence similarity to the HlyA signal sequence, and we have hypothesized that these signal sequences assume a similar higher-order structure which is recognized by the HlyB/D transporter. In the present study, we have expressed and purified small peptides containing the C-terminal 61 amino acids of HlyA and the C-terminal 70 amino acids of LktA. We show that these signal peptides are sufficient for secretion from E. coli in a HlyB/D dependent manner. Circular dichroism analyses show that both molecules exhibit common biophysical properties. In aqueous solution, they appear to be mainly unstructured, but in a membrane mimetic environment they assume a helical secondary structure. The conformational change observed for both peptides going from an aqueous to a membrane mimetic environment may be an important feature of these signal sequences necessary for their recognition and transport.

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“…The initial recognition of an uncleaved C-terminal secretory signal of RTX toxin by the ABC transporter and MFP triggers the assembly of a functional trans-envelope complex through further specific interactions of MFP and OMP in the periplasm. This transport complex bypasses the periplasm and releases the toxin directly into the extracellular environment in a single step mechanism ( Figure 6 ) [ 190 , 209 , 210 , 211 , 212 , 213 , 214 ]. Interestingly, the function of the ABC and MFP proteins is unique to the T1SS, whereas TolC is pleiotropic and has a variety of other functions, such as efflux of toxic molecules [ 215 , 216 ].…”

Section: Secretion Of Rtxa and Other Rtx Toxinsmentioning

confidence: 99%

“…The ABC transporter of uropathogenic E. coli called HlyB is a 77 kDa protein located in the inner bacterial membrane [ 212 , 220 ] and is thought to be active as a homodimer [ 209 , 212 , 217 , 218 , 220 ]. HlyB consists of an N-terminal cytoplasmic domain, a transmembrane domain (TMD) that anchors the protein in the inner membrane, and a C-terminal nucleotide binding domain (NBD) in the cytoplasm [ 209 , 221 , 222 ].…”

Section: Secretion Of Rtxa and Other Rtx Toxinsmentioning

confidence: 99%

Kingella kingae RtxA Cytotoxin in the Context of Other RTX Toxins

et al. 2022

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The Gram-negative bacterium Kingella kingae is part of the commensal oropharyngeal flora of young children. As detection methods have improved, K. kingae has been increasingly recognized as an emerging invasive pathogen that frequently causes skeletal system infections, bacteremia, and severe forms of infective endocarditis. K. kingae secretes an RtxA cytotoxin, which is involved in the development of clinical infection and belongs to an ever-growing family of cytolytic RTX (Repeats in ToXin) toxins secreted by Gram-negative pathogens. All RTX cytolysins share several characteristic structural features: (i) a hydrophobic pore-forming domain in the N-terminal part of the molecule; (ii) an acylated segment where the activation of the inactive protoxin to the toxin occurs by a co-expressed toxin-activating acyltransferase; (iii) a typical calcium-binding RTX domain in the C-terminal portion of the molecule with the characteristic glycine- and aspartate-rich nonapeptide repeats; and (iv) a C-proximal secretion signal recognized by the type I secretion system. RTX toxins, including RtxA from K. kingae, have been shown to act as highly efficient ‘contact weapons’ that penetrate and permeabilize host cell membranes and thus contribute to the pathogenesis of bacterial infections. RtxA was discovered relatively recently and the knowledge of its biological role remains limited. This review describes the structure and function of RtxA in the context of the most studied RTX toxins, the knowledge of which may contribute to a better understanding of the action of RtxA in the pathogenesis of K. kingae infections.

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Characterization of the hemolysin transporter, HlyB, using an epitope insertion.

Cited by 34 publications

References 26 publications

Phosphatidylcholine and Phosphatidylethanolamine Behave as Substrates of the Human MDR1 P-Glycoprotein

Phosphatidylcholine and Phosphatidylethanolamine Behave as Substrates of the Human MDR1 P-Glycoprotein

Secretion and Circular Dichroism Analysis of the C-Terminal Signal Peptides of HlyA and LktA

Kingella kingae RtxA Cytotoxin in the Context of Other RTX Toxins

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