Effects of serum carrier proteins on the growth of pathogenic neisseriae with heme-bound iron.

The pathogenic neisseriae can use free heme and hemoglobin as an essential source of iron (Fe) for growth in vitro, but it is unknown whether they can utilize heme bound to human hemopexin or to human serum albumin, or hemoglobin bound to haptoglobin. We found that neither Neisseria meningitidis nor Neisseria gonorrhoeae used bound heme, but bound hemoglobin was used as an Fe source by two meningococcal strains and one gonococcal strain. A second gonococcal strain, previously shown to use free hemoglobin poorly or not at all, also did not grow with hemoglobin-haptoglobin complex as an Fe source. These observations suggest that hemoglobin might act as an Fe source in vivo for many pathogenic neisseriae even when in complexed (bound) form, but heme probably would not support growth in vivo if bound to serum carrier proteins.     

Identification and purification of a hemoglobin-binding outer membrane protein from Neisseria gonorrhoeae.

The majority of in vitro-grown Neisseria gonorrhoeae strains were unable to use hemoglobin as the sole source of iron for growth (Hgb-), but a minor population was able to do so (Hgb+). The ability of Hgb+ gonococci to utilize hemoglobin as the iron source was associated with the expression of an iron-repressible 89-kDa hemoglobin-binding protein in the outer membrane. The N-terminal amino acid sequence of this protein revealed amino acids, from positions 2 to 16, identical to those of HpuB, an 85 kDa iron-regulated hemoglobin-haptoglobin utilization outer membrane protein of Neisseria meningitidis. Isogenic mutants constructed by allelic replacement with a meningococcal hpu::mini-Tn3erm construct no longer expressed the 89-kDa protein. Mutants could not utilize hemoglobin to support growth but still grew on heme. Thus, the gonococcal HpuB homolog is a functional hemoglobin receptor and is essential for growth with hemoglobin.     

exl, an exchangeable genetic island in Neisseria meningitidis

The genetic structure and evolution of a novel exchangeable meningococcal genomic island was defined for the important human pathogen Neisseria meningitidis. In 125 meningococcal strains tested, one of three unrelated nucleotide sequences, designated exl (exchangeable locus), was found between a gene required for heme utilization, hemO, and col, encoding a putative Escherichia coli collagenase homologue. The 5' boundary of each exl cassette was the stop codon of hemO, whereas the 3' boundary was delineated by a 33-bp repeat containing neisserial uptake sequences located downstream of col. One of the three alternative exl cassettes contained the meningococcal hemoglobin receptor gene, hmbR (exl3). In other meningococcal strains, hmbR was absent from the genome and was replaced by either a nucleotide sequence containing a novel open reading frame, exl2, or a cassette containing exl3. The proteins encoded by exl2 and exl3 had no significant amino acid homology to HmbR but contained six motifs that are also present in the lipoprotein components of the lactoferrin (LbpB), transferrin (TbpB), and hemoglobin-haptoglobin (HpuA) uptake systems. To determine the evolutionary relationships among meningococci carrying hmbR, exl2, or exl3, isolates representing 92 electrophoretic types were examined. hmbR was found throughout the population structure of N. meningitidis (genetic distance, >0.425), whereas exl2 and exl3 were found in clonal groups at genetic distances of <0.2. The commensal neisserial species were identified as reservoirs for all of the exl cassettes found in meningococci. The structure of these cassettes and their correlation with clonal groups emphasize the extensive gene pool and frequent horizontal DNA transfer events that contribute to the evolution and virulence of N. meningitidis.     

Identification of an outer membrane protein involved in utilization of hemoglobin-haptoglobin complexes by nontypeable Haemophilus influenzae.

A recombinant plasmid containing a 6.5-kb fragment of nontypeable Haemophilus influenzae (NTHI) chromosomal DNA was shown to confer a hemoglobin-haptoglobin-binding phenotype on Escherichia coli. Use of a mini-Tn10kan transposon for random insertion mutagenesis of this recombinant plasmid allowed localization of the NTHI DNA responsible for this hemoglobin-haptoglobin-binding phenotype to a 3.5-kb PstI-XhoI fragment within the 6.5-kb NTHI DNA insert. When this mutagenized NTHI DNA fragment was used to transform the wild-type NTHI strain, the resultant kanamycin-resistant mutant exhibited significantly decreased abilities to bind hemoglobin-haptoglobin and utilize it as a source of heme for aerobic growth in vitro. This mutant also lacked expression of a 115-kDa outer membrane protein that was present in the wild-type parent strain. Transformation of this mutant with wild-type NTHI chromosomal DNA restored the abilities to bind and utilize hemoglobin-haptoglobin and to express the 115-kDa outer membrane protein. Nucleotide sequence analysis of the relevant NTHI DNA revealed the presence of a gene, designated hhuA, that encoded a predicted 117,145-Da protein. The HhuA protein exhibited features typical of a TonB-dependent outer membrane receptor and had significant identity with the hemoglobin receptors of both Haemophilus ducreyi and Neisseria meningitidis.     

Identification and characterization of a Streptococcus pyogenes operon involved in binding of hemoproteins and acquisition of iron

The hemolytic Streptococcus pyogenes can use a variety of heme compounds as an iron source. In this study, we investigate hemoprotein utilization by S. pyogenes. We demonstrate that surface proteins contribute to the binding of hemoproteins to S. pyogenes. We identify an ABC transporter from the iron complex family named sia for streptococcal iron acquisition, which consists of a lipoprotein (siaA), membrane permease (siaB), and ATPase (siaC). The sia transporter is part of a highly conserved, iron regulated, 10-gene operon. SiaA, which was localized to the cell membrane, could specifically bind hemoglobin. The operon's first gene encodes a novel bacterial protein that bound hemoglobin, myoglobin, heme-albumin, and hemoglobin-haptoglobin (but not apo-haptoglobin) and therefore was named Shr, for streptococcal hemoprotein receptor. PhoZ fusion and Western blot analysis showed that Shr has a leader peptide and is found in both membrane-bound and soluble forms. An M1 SF370 strain with a polar mutation in shr was more resistant to streptonigrin and hydrogen peroxide, suggesting decreased iron uptake. The addition of hemoglobin to the culture medium increased cell resistance to hydrogen peroxide in SF370 but not in the mutant, implying the sia operon may be involved in hemoglobin-dependent resistance to oxidative stress. The shr mutant demonstrated reduced hemoglobin binding, though cell growth in iron-depleted medium supplemented with hemoglobin, whole blood, or ferric citrate was not affected, suggesting additional systems are involved in hemoglobin utilization. SiaA and Shr are the first hemoprotein receptors identified in S. pyogenes; their possible role in iron capture is discussed.     

A sensitive solid-phase assay for identification of class A macrophage scavenger receptor ligands using cell lysate

In order for macrophages to perform their numerous homeostatic, immunological and tissue remodeling functions they are required to express a broad repertoire of cell-surface receptors. These receptors are particularly important for their host-defense functions in the recognition of foreign pathogens. Delineation of the particular functions of specific receptors requires the identification of ligands recognized by the receptor. We have developed a sensitive, high throughput, solid-phase assay for the detection of ligands for the class A macrophage scavenger receptor (SR-A). Post-nuclear cell lysate from murine bone marrow-derived macrophages is used as a source of receptor and specific ligand binding to SR-A is detected with a monoclonal antibody for SR-A. This assay has been used effectively to identify protein ligands for SR-A on the surface of the bacterium Neisseria meningitidis (Peiser, L. et al. [Peiser, L., Makepeace, K., Pluddemann, A., Savino, S., Wright, J.C., Pizza, M., Rappuoli, R., Moxon, E.R., Gordon, S., 2006. Identification of Neisseria meningitidis nonlipopolysaccharide ligands for class A macrophage scavenger receptor by using a novel assay. Infect. Immun. 74, 5191-5199]). In this paper we describe the method in detail and define the specific variables governing the assay.     

Phase Variation of Hemoglobin Utilization in Neisseria gonorrhoeae

Most Neisseria gonorrhoeae isolates are unable to use human hemoglobin as the sole source of iron for growth (Hgb⁻), but a minor population is able to do so (Hgb⁺). This minor population grows luxuriously on hemoglobin, expresses two outer membrane proteins of 42 kDa (HpuA) and 89 kDa (HpuB), and binds hemoglobin under iron-stressed conditions. In addition to the previously reported HpuB, we identified and characterized HpuA, which is encoded by the gene hpuA, located immediately upstream of hpuB. Expression of both proteins was found to be controlled at the translational level by frameshift mutations in a run of guanine residues within the hpuA sequence encoding the mature HpuA protein. The “on-phase” hemoglobin-utilizing variants contained 10 G’s, while the “off-phase” variants contained 9 G’s. Insertional hpuB mutants of FA19 Hgb⁺ and FA1090 Hgb⁺ no longer expressed HpuB but still produced HpuA. A polar insertional mutation of the upstream hpuA gene in FA1090 Hgb⁺ eliminated production of both HpuA and HpuB, whereas a nonpolar insertional mutant expressed HpuB only. Insertional mutagenesis of either hpuA or hpuB or both substantially decreased the hemoglobin binding ability of the FA1090 Hgb⁺ variant and prevented growth on hemoglobin plates. Therefore, both HpuA and HpuB were required for the utilization of hemoglobin for growth.     

Effect of multiple mutations in the hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae type b.

Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA and hgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approximately 120 kDa from the hgpA hgpB double mutant. We have cloned and sequenced the gene encoding this third hemoglobin/hemoglobin-haptoglobin binding protein and designate it hgpC. Insertional mutation of hgpC did not affect the ability of the strain to utilize either hemoglobin or hemoglobin-haptoglobin. An hgpA hgpB hgpC triple mutant constructed by insertional mutagenesis showed a reduced ability to use the hemoglobin-haptoglobin complex but was unaltered in the ability to use hemoglobin. A second class of mutants was constructed in which the entire structural gene of each of the three proteins was deleted. The hgpA hgpB hgpC complete-deletion triple mutant was unable to utilize the hemoglobin-haptoglobin complex and showed a reduced ability to use hemoglobin. We have identified three hemoglobin/hemoglobin-haptoglobin-binding proteins in Haemophilus influenzae. Any one of the three proteins is sufficient to support growth with hemoglobin-haptoglobin as the heme source, and expression of at least one of the three is essential for hemoglobin-haptoglobin utilization. Although the three proteins play a role in hemoglobin utilization, an additional hemoglobin acquisition mechanism(s) exists.     

Neisseria gonorrhoeae Heme Biosynthetic Mutants Utilize Heme and Hemoglobin as a Heme Source but Fail To Grow within Epithelial Cells

Many bacterial pathogens, including pathogenic neisseriae, can use heme as an iron source for growth. To study heme utilization by Neisseria gonorrhoeae, two heme biosynthetic mutants were constructed, one with a mutation in hemH (the gene encoding ferrochelatase) and one with a mutation in hemA (the gene encoding γ-glutamyl tRNA reductase). The hemH mutant failed to grow without an exogenous supply of heme or hemoglobin, whereas the hemA mutant failed to grow unless heme, hemoglobin, or heme precursors were present. Growth of the mutants with hemoglobin required expression of the hemoglobin receptor (HpuAB) and was TonB dependent. However, growth with heme required neither HpuAB nor TonB. An fbpA mutant grew normally when either heme or hemoglobin was present in the medium. The heme biosynthetic mutants showed reduced intracellular survival, compared to the parent strain, within A-431 endocervical epithelial cell cultures. These studies demonstrate that in addition to synthesizing their own heme, N. gonorrhoeae strains are able to internalize and utilize exogenous heme independently of FbpA but appear unable to obtain heme from within epithelial cells for growth.     

Plasma protein haptoglobin modulates renal iron loading

Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. The strength of hemoglobin binding and the existence of a specific receptor for the haptoglobin-hemoglobin complex in the monocyte/macrophage system clearly suggest that haptoglobin may have a crucial role in heme-iron recovery. We used haptoglobin-null mice to evaluate the impact of haptoglobin gene inactivation on iron metabolism. Haptoglobin deficiency led to increased deposition of hemoglobin in proximal tubules of the kidney instead of the liver and the spleen as occurred in wild-type mice. This difference in organ distribution of hemoglobin in haptoglobin-deficient mice resulted in abnormal iron deposits in proximal tubules during aging. Moreover, iron also accumulated in proximal tubules after renal ischemia-reperfusion injury or after an acute plasma heme-protein overload caused by muscle injury, without affecting morphological and functional parameters of renal damage. These data demonstrate that haptoglobin crucially prevents glomerular filtration of hemoglobin and, consequently, renal iron loading during aging and following acute plasma heme-protein overload.     

Reduced severity of middle ear infection caused by nontypeable Haemophilus influenzae lacking the hemoglobin/hemoglobin-haptoglobin binding proteins (Hgp) in a chinchilla model of otitis media

Since Haemophilus influenzae lacks enzymes necessary for synthesis of the porphyrin ring, it has an absolute growth requirement for a porphyrin source. This requirement can be satisfied in vitro by hemoglobin and hemoglobin complexed to haptoglobin. The products of the hgp genes mediate the utilization of heme from hemoglobin-haptoglobin. These genes are also involved in the use of heme from hemoglobin, although additional gene products independently mediate the acquisition of heme from this substrate. Different strains of H. influenzae possess one to four hgp genes. A nontypeable H. influenzae mutant lacking all the hgp genes was constructed and compared to the wild-type strain in a chinchilla (Chinchilla lanigera) model of otitis media. Compared to the wild-type strain, the hgp-deficient mutant exhibited a significantly delayed onset of detectable middle ear infection and significantly reduced duration of infection as assessed by both video otoscopy and tympanometry and as evidenced by viable bacterial counts in middle ear effusions. In addition, the maximum bacterial load in the middle ears of chinchillas infected with the mutant strain was significantly reduced when compared to the parent. These data indicate that the hemoglobin/hemoglobin-haptoglobin binding proteins are required for bacterial proliferation during H. influenzae-induced otitis media in chinchillas.     

The acute-phase reactant C-reactive protein binds to phosphorylcholine-expressing Neisseria meningitidis and increases uptake by human phagocytes

Neisseria meningitidis is a global cause of meningitis and septicemia. Immunity to N. meningitidis involves both innate and specific mechanisms with killing by serum bactericidal activity and phagocytic cells. C-reactive protein (CRP) is an acute-phase serum protein that has been shown to help protect the host from several bacterial pathogens, which it recognizes by binding to phosphorylcholine (PC) on their surfaces. Pathogenic Neisseria species can exhibit phase-variable PC modification on type 1 and 2 pili. We have shown that CRP can bind to piliated meningococci in a classical calcium-dependent manner. The binding of CRP to the meningococcus was concentration dependent, of low affinity, and specific for PC. CRP appears to act as an opsonin for N. meningitidis, as CRP-opsonized bacteria showed increased uptake by human macrophages and neutrophils. Further investigation into the downstream effects of CRP-bound N. meningitidis may lead us to a better understanding of meningococcal infection and help direct more effective therapeutic interventions.     

A general affinity chromatographic method for preparing monospecific antibody to mammalian serum haptoglobin

A general affinity chromatographic method for preparation of monospecific antibody to serum haptoglobin of any species is described. Hemoglobin prepared from the species to be immunized is coupled to an organomercurical substituted agarose gel support (Affi-Gel 501). The immobilized hemoglobin binds haptoglobin with great affinity and allows removal of other serum proteins by extensive washing. The haptoglobin-hemoglobin complexes are then specifically eluted by buffers containing dithiothreitol or other thiols and are further purified by chromatography on concanavalin A-agarose and Sephacryl S-200 columns. The pure complexes are very effective immunogens. Potent monospecific antisera to rabbit and to human haptoglobin have been prepared. The potential usefulness of affinity chromatography support media with specifically cleavable ligand sites in studies of haptoglobin and in other biological studies is discussed.     

Protein sources of heme for Haemophilus influenzae.

Although Haemophilus influenzae requires heme for growth, the source of heme during invasive infections is not known. We compared heme, lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin as sources of heme for growth in defined media. The minimum concentration of heme permitting unrestricted growth of strain E1a, an H. influenzae type b isolate from cerebrospinal fluid, was 0.02 micrograms/ml. Using molar equivalents of heme as lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin, we determined that myoglobin and hemoglobin permitted unrestricted growth at this concentration. To determine the ability of host defenses to sequester heme from H. influenzae, we used affinity chromatography to purify human haptoglobin and hemopexin, serum proteins which bind hemoglobin and heme. Plate assays revealed that 12 strains of H. influenzae acquired heme from hemoglobin, hemoglobin-haptoglobin, heme-hemopexin, and heme-albumin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of strain E1a grown in heme-replete and heme-restricted conditions revealed a heme-repressible outer membrane protein with an apparent molecular mass of 38 kilodaltons. These results demonstrated that, unlike Escherichia coli, H. influenzae may acquire heme from hemoglobin-haptoglobin. H. influenzae also may acquire heme from hemopexin and albumin, which have not been previously investigated. The role of outer membrane proteins in the acquisition of heme is not yet clear.     

Human airway epithelial cell responses to Neisseria lactamica and purified porin via Toll-like receptor 2-dependent signaling

The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin's surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.     

Quantitative aspects of ouabain binding to human erythrocyte and cardiac membranes.

1. [3H]ouabain binding to human erythrocyte membranes is a time- and temperature-dependent process. The association of ouabain to the membrane-bound receptor follows second-order kinetics, while the dissociation is a monomolecular reaction. An association rate constant of 4-6 x 10(4) M-1 sec-1 and a dissociation rate constant of 1-4 x 10(-4) sec-1 were measured at 37 degrees C. The dissociation constant calculated from these data agrees with that determined from equilibrium binding experiments. There is only one type of ouabain binding sites with high affinity for the drug as reflected by the low dissociation constant of 0-28 x 10(-8) M. 2. The dissociation constants of the ouabain-receptor complexes from human erythrocyte and cardiac membranes are identical. 3. The maximal number of membrane-bound ouabain binding sites was measured from equilibrium binding experiments as 288 +/- 28 per single erythrocyte. Thus one receptor site corresponds to less than 1 mum2 of the membrane, provided the receptors are diffusely distributed on the surface of the membrane. 4. Neither the maximal number of ouabain receptors nor the affinity for the drug changes with the age or sex of the blood donor. 5. A maximal transport capacity for sodium of 5-6 m-equiv/hr.1. is calculated from the number of receptor sites per erythrocyte and from the turn-over number of the (Na+ + K+)-ATPase.     

Characterization of Neisseria meningitidis isolates that do not express the virulence factor and vaccine antigen factor H binding protein

Neisseria meningitidis remains a leading cause of bacterial sepsis and meningitis. Complement is a key component of natural immunity against this important human pathogen, which has evolved multiple mechanisms to evade complement-mediated lysis. One approach adopted by the meningococcus is to recruit a human negative regulator of the complement system, factor H (fH), to its surface via a lipoprotein, factor H binding protein (fHbp). Additionally, fHbp is a key antigen in vaccines currently being evaluated in clinical trials. Here we characterize strains of N. meningitidis from several distinct clonal complexes which do not express fHbp; all strains were recovered from patients with disseminated meningococcal disease. We demonstrate that these strains have either a frameshift mutation in the fHbp open reading frame or have entirely lost fHbp and some flanking sequences. No fH binding was detected to other ligands among the fHbp-negative strains. The implications of these findings for meningococcal pathogenesis and prevention are discussed.     

Oxygen-linked CO2 transport in sheep blood.

We have analyzed oxygen-linked carbamate formation in sheep hemoglobin B by measuring a) the effect of CO2 on oxygen affinity and Bohr effect in red cell suspensions and dilute (1.3 mM Hb4) and concentrated (5 mM Hb4) hemoglobin solutions at 37 degrees C and b) CO2 binding curves of deoxygenated and oxygenated whole blood and hemoglobin solutions, respectively, at the same temperature. In the presence of CO2 both the Bohr effect and oxygen affinity were significantly lower in 1.3-mM Hb4 solutions than in either red cell suspensions or 5-mM Hb4 solutions, while in the absence of CO2 Bohr effect and oxygen affinity did not differ significantly in those preparations. Likewise, the fraction of oxygen-linked carbamate obtained from CO2 binding curves was found to be higher in 1.3-mM Hb4 (0.156 M HbCO2/M HbO2) solutions than in 5-mM Hb4 solutions (0.12 M HbCO2/M HbO2) at pH 7.2. We conclude that hemoglobin concentration affects formation of oxygen-linked carbamate. Total oxygen-linked CO2 in sheep whole blood amounted to 0.18 M CO2/M O2 of which 70% is oxygen-linked carbamate. Assuming a respiratory quotient of 0.85, the contribution of oxygen-linked CO2 to carbon dioxide exchange in sheep blood was computed to be 21%.     

Identification of Neisseria meningitidis nonlipopolysaccharide ligands for class A macrophage scavenger receptor by using a novel assay

Macrophages (Mphi) may play an important role in the pathogenesis of invasive meningococcal infection. Previously, we have shown that the class A Mphi scavenger receptor (SR-A) is a major nonopsonic receptor for Neisseria meningitidis on Mphi. SR-A contributes to host defense by binding proinflammatory polyanionic ligands such as lipopolysaccharide (LPS) and by the uptake and killing of live organisms. SR-A-deficient mouse Mphi display a substantial reduction in the number of meningococci ingested compared to wild-type Mphi, and SR-A is required for meningococcal phagocytosis but not for the release of tumor necrosis factor alpha. Although soluble lipid A and lipid(IV)A are reported as ligands for SR-A, we demonstrated that LPS and LPS expression were not essential for the uptake of whole meningococci. In the present study, we set out to discover protein ligand(s) for SR-A in N. meningitidis lysates and outer membrane vesicles. Using various microbial mutant strains, we determined that molecules comprising the membrane capsule and pili, as well as the abundant surface Opa proteins were not essential for SR-A recognition. We developed a binding assay to detect SR-A ligands and identified three candidate proteins expressed on intact organisms, namely, NMB1220, NMB0278, and NMB0667. Soluble forms of these ligands were shown to block the binding of meningococci to CHO cells stably transfected with SR-A. Furthermore, NMB1220 was endocytosed by SR-A on Mphi and prevented internalization of soluble acetylated low-density lipoprotein. Thus, we have identified novel, unmodified protein ligands for SR-A that are able to inhibit meningococcal interactions with macrophages in vitro.     

Target specificity of selective bioactive compounds in blocking α-dystroglycan receptor to suppress Lassa virus infection: an in silico approach

Lassa hemorrhagic fever, caused by Lassa mammarenavirus (LASV) infection, accumulates up to 5000 deaths every year. Currently, there is no vaccine available to combat this disease. In this study, a library of 200 bioactive compounds was virtually screened to study their drug-likeness with the capacity to block the α-dystroglycan (α-DG) receptor and prevent LASV influx. Following rigorous absorption, distribution, metabolism, and excretion (ADME) and quantitative structure-activity relationship (QSAR) profiling, molecular docking was conducted with the top ligands against the α-DG receptor. The compounds chrysin, reticuline, and 3-caffeoylshikimic acid emerged as the top three ligands in terms of binding affinity. Post-docking analysis revealed that interactions with Arg76, Asn224, Ser259, and Lys302 amino acid residues of the receptor protein were important for the optimum binding affinity of ligands. Molecular dynamics simulation was performed comprehensively to study the stability of the protein-ligand complexes. In-depth assessment of root-mean-square deviation (RMSD), root mean square fluctuation (RMSF), polar surface area (PSA), B-Factor, radius of gyration (Rg), solvent accessible surface area (SASA), and molecular surface area (MolSA) values of the protein-ligand complexes affirmed that the candidates with the best binding affinity formed the most stable protein-ligand complexes. To authenticate the potentialities of the ligands as target-specific drugs, an in vivo study is underway in real time as the continuation of the research.