Biology subjects

Khatoon, N.

Publications and source records attributed to Khatoon, N..

4 recordsLinked to original sources

bioRxiv2025

Parenteral Vaccination with recombinant EtpA glycoprotein impairs enterotoxigenic E. coli colonization

Enterotoxigenic E. coli (ETEC) cause hundreds of millions of cases of acute diarrheal illness in low-middle income regions, disproportionately in young children. To date there is no licensed, broadly protective vaccine to protect against these common but antigenically heterogeneous pathogens. One of the more highly conserved antigens of ETEC, EtpA, is an extracellular glycoprotein adhesin that preferentially binds to blood group A glycans on intestinal epithelia. EtpA contributes to increased severity of illness in blood group A individuals, elicits robust serologic and fecal antibody responses following infection, and has been associated with protection against subsequent infection. However, its utility as a protective antigen needs further examination. In the present studies we examined whether parenteral vaccination with recombinant EtpA (rEtpA) could afford protection against intestinal colonization in a murine model of ETEC infection. Here, we demonstrate that intramuscular vaccination with rEtpA when adjuvanted with double mutant LT (dmLT) primes IgG predominant mucosal antibody responses to ETEC challenge. Notably, however, both antibody levels and avidity, as well as protection were dependent on vaccination schedule. Likewise, by electron microscopy polyclonal epitope mapping (EMPEM) we observed a greater diversity of epitopes targeted by antibodies after a more protracted vaccination schedule. Next, we explored the utility of IM immunization with alum-adjuvanted rEtpA. This elicited strong serologic and fecal IgG responses. Although accompanied by negligible IgA mucosal responses, EtpA alum-adjuvanted IM vaccination nevertheless protected against ETEC intestinal colonization. Collectively, these data suggest that EtpA could expand the portfolio of antigens targeted in ETEC subunit vaccine development.

microbiology↗

bioRxiv2024

Role of surface negative charges in agonist binding to the unliganded open state of the neuromuscular acetylcholine receptor

Acetylcholine receptors (AChRs) expressed at the nerve muscle junction (NMJ) synapses are hetero-pentameric ligand-gated ion channels with two neurotransmitter binding sites (TBS) at -{delta} and -{varepsilon} (adult)/{gamma} (fetal) subunit interfaces. They are typical allosteric proteins which reside in at least two stable conformations: resting (Closed) and active (Open) states. Mechanism of agonist (A) binding to the C state has been extensively studied, but agonist association to the O state has not been clearly understood. Here, by using engineered constitutively active AChRs, single-channel patch-clamp, and molecular dynamics (MD) simulations, we elucidate the differences between unliganded (O) vs liganded (AO) active states and the mechanism of agonist association to the O state. Our results indicate that: 1. At the {gamma}-binding site, for a series of agonists, the O state agonist association rate (jon) was [~]30 times faster than the diffusion limit and agonist affinity was 2-orders of magnitude higher vs at the {delta}- and {varepsilon}-binding sites. 2. Electrostatic surface charge density owing to 4 negatively charged residues ({gamma}E57, {gamma}D113, {gamma}D174, and {gamma}E180) facilitates loop C capping, compaction of the TBS, and agonist stabilization in the AO state. 3. Mutating these residues in combination reversed the jon and binding affinity to those comparable to the {delta}- and {varepsilon}-binding sites. 4. {varphi}-value analysis indicated the presence of a transition state intermediate between the O and AO states. Overall, we elucidate the role of neighboring charged residues outside the TBS in determining high-affinity agonist binding and their significance in shaping the synaptic response at the NMJ. SummaryNicotinic acetylcholine receptors (AChRs) are allosteric proteins that are crucial for muscle contraction. The isomerization of resting closed (C) to active open (O) state can be achieved by both bind-gate and gate-bind (rare) pathway. Little is known about agonist binding to the unliganded functional O state. Here, we elucidate the nature of unliganded O state and the mechanism of agonist binding to this high-affinity O state by single-channel current recordings, protein engineering, in silico methods and phi-analysis. Our results describe an energetic barrier between the unliganded and liganded O state owing to negatively charged amino acids near the agonist site. These charges partially contribute to the low- and high-affinity agonist energies specifically at the embryonic neurotransmitter binding site. We also discussed the physiological significance of these residues in shaping synaptic response. Significance StatementEmbryonic-type AChRs are indispensable for nerve-muscle junction formation. Many mutations in these receptors cause congenital myasthenia syndrome by increasing constitutive channel activation. Therefore, understanding the nature of unliganded functional open (O) state is critical. Here, we describe the nature and the mechanism of agonist binding to the unliganded O (or apo) state. Single-channel currents were recorded with different background constructs and binding site mutations. The results highlight the importance of loop C and ligand orientation in determining high-affinity agonist binding. Further we elucidate the role of electrostatic interactions in agonist binding to the O state (high-affinity binding). We surmise that higher association rate of agonist binding to O state is the reason behind higher efficacy of the fetal receptor.

biophysics↗

bioRxiv2024

Host-derived CEACAM-laden vesicles engage enterotoxigenic E. coli for elimination and toxin neutralization.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of diarrheal illnesses annually ranging from mildly symptomatic cases to severe, life-threatening cholera-like diarrhea. Although ETEC are associated with long-term sequelae including malnutrition, the acute diarrheal illness is largely self-limited. Recent studies indicate that in addition to causing diarrhea, the ETEC heat-labile toxin (LT) modulates the expression of many genes in intestinal epithelia, including carcinoembryonic cell adhesion molecules (CEACAMs) which ETEC exploit as receptors, enabling toxin delivery. Here however, we demonstrate that LT also enhances the expression of CEACAMs on extracellular vesicles (EV) shed by intestinal epithelia and that CEACAM-laden EV increase in abundance during human infections, mitigate pathogen-host interactions, scavenge free ETEC toxins, and accelerate ETEC clearance from the gastrointestinal tract. Collectively, these findings indicate that CEACAMs play a multifaceted role in ETEC pathogen-host interactions, transiently favoring the pathogen, but ultimately contributing to innate responses that extinguish these common infections. Significance statementEnterotoxigenic E. coli, characterized by the production of heat-labile (LT) and heat-stable (ST) toxins, are a very common cause of diarrhea in low-income regions responsible for hundreds of millions of infections each year, and the major cause of diarrhea in travelers to endemic areas. Although these infections may be severe and cholera-like, they are typically self-limited. These studies demonstrate that extracellular vesicles produced by host intestinal cells can capture the bacteria and its secreted toxins at a distance from the cell surface, potentially acting as molecular decoys to neutralize the enterotoxins and extinguish the infection.

microbiology↗

bioRxiv2024

Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as human volunteers challenged with ETEC, diarrheal severity is significantly increased severity in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of human volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected human volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design. Author summaryEnterotoxigenic E. coli (ETEC), a leading cause of diarrhea disproportionately affecting young children in low-income regions, are a priority for vaccine development. Individuals possessing A blood-type are more susceptible to severe cholera-like disease. EtpA, a secreted, immunogenic, blood group A binding protein, is a current vaccine target antigen. Here, we determined the atomic structure of EtpA in complex with protective as well as non-protective monoclonal antibodies targeting two different domains of the protein, allowing us to pinpoint key regions involved in blood-group A antigen recognition and uncover the mechanism of antibody-based protection. In addition, we show through mass-spectrometry that EtpA is extensively and heterogeneously glycosylated at surface-exposed asparagine residues by a promiscuous and low-fidelity glycosyltransferase, EtpC, and that this unique form of bacterial glycosylation is critical for to development of protective immune responses. Lastly, polyclonal antibodies from vaccinated mice as well as monoclonal antibodies obtained from ETEC-infected human volunteers revealed that the highly antigenic surface of EtpA exhibits both protective and non-protective epitopes. These results greatly expand our understanding of ETEC pathogenesis, and the immune responses elicited by these common infections, providing valuable information to aid in the rational design and testing of subunit vaccines.

microbiology↗