A substantial disease burden in vertebrates is due to Gram-negative bacteria that exclusively inhabit the upper respiratory or genitourinary tracts of their hosts and rely on directly acquiring iron ...from the host iron-binding glycoproteins through surface receptor proteins. The receptors enable these bacteria to proliferate independently from their neighbors on the mucosal surface and during invasive infection of the host. The diversity in these receptors evolved over millions of years of evolution, which thus bodes well for long-lasting vaccine coverage. Experiments in food production animals provide proof of concept for the use of engineered antigens derived from the receptor proteins to prevent colonization and invasive infection in the natural host, strongly supporting development of these vaccines for use in humans.
The presence of transferrin (Tf) receptors in Gram-negative bacteria that reside in the upper respiratory tract of mammals and birds indicate that they have existed for over 320 million years.Tf/lactoferrin receptors are essential for survival of Gram-negative bacterial pathogens (pathobionts) on the mucosal surface and during invasive infection and thus are ideal targets for vaccines.Structure-based antigen engineering can improve the immune response induced by Tf receptor proteins.Proof of concept experiments in pigs demonstrate that antigens targeting Tf receptors can prevent infection and colonization.
Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity ...among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein's C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Moraxella catarrhalis
is a Gram-negative bacterium that is responsible for a substantial proportion of upper respiratory infections in children and lower respiratory infections in the elderly.
...Moraxella catarrhalis
resides exclusively on the mucosal surfaces of the upper respiratory tract of humans and is capable of directly acquiring iron for growth from the host glycoproteins human transferrin (hTf) and human lactoferrin (hLf). The iron-bound form of these glycoproteins is initially captured by the surface lipoproteins Tf or Lf binding protein B (TbpB or LbpB) and delivered to the integral outer membrane TonB-dependent transport (TBDT) proteins, Tf binding protein A (TbpA) or Lf binding protein A (LbpA). The extraction of iron involves conformational changes in Lf and Tf to facilitate iron removal followed by its transport across the outer membrane by a well characterized process for TBDTs. Surprisingly the disruption of the gene encoding another TBDT, CopB, results in a reduction in the ability to grow on human Tf or Lf. The possibility that this could have been due to an artifact of mutant construction that resulted in the inhibition of TonB-mediated process was eliminated by a complete deletion of the CopB gene. A systematic evaluation of the impact on growth under various conditions by deletions of the genes encoding TbpA, LbpA, and CopB as well as mutations of the iron liganding residues and TonB box region of CopB was implemented. The results indicate that although CopB is capable of effectively acquiring iron from the growth medium, it does not directly acquire iron from Tf or Lf. We propose that the indirect effect on iron transport from Tf and Lf by CopB could possibly be explained by the association of TBDTs at gaps in the peptidoglycan layer that may enhance the efficiency of the process. This concept is supported by previous studies demonstrating an indirect effect on growth of Tf and Lf by deletion of the peptidoglycan binding outer membrane lipoprotein RmpM in
Neisseria
that also reduced the formation of larger complexes of TBDTs.
Iron is an essential element for various lifeforms but is largely insoluble due to the oxygenation of Earth’s atmosphere and oceans during the Proterozoic era. Metazoans evolved iron transport ...glycoproteins, like transferrin (Tf) and lactoferrin (Lf), to keep iron in a non-toxic, usable form, while maintaining a low free iron concentration in the body that is unable to sustain bacterial growth. To survive on the mucosal surfaces of the human respiratory tract where it exclusively resides, the Gram-negative bacterial pathogen
Moraxella catarrhalis
utilizes surface receptors for acquiring iron directly from human Tf and Lf. The receptors are comprised of a surface lipoprotein to capture iron-loaded Tf or Lf and deliver it to a TonB-dependent transporter (TBDT) for removal of iron and transport across the outer membrane. The subsequent transport of iron into the cell is normally mediated by a periplasmic iron-binding protein and inner membrane transport complex, which has yet to be determined for
Moraxella catarrhalis
. We identified two potential periplasm to cytoplasm transport systems and performed structural and functional studies with the periplasmic binding proteins (FbpA and AfeA) to evaluate their role. Growth studies with strains deleted in the
fbpA
or
afeA
gene demonstrated that FbpA, but not AfeA, was required for growth on human Tf or Lf. The crystal structure of FbpA with bound iron in the open conformation was obtained, identifying three tyrosine ligands that were required for growth on Tf or Lf. Computational modeling of the YfeA homologue, AfeA, revealed conserved residues involved in metal binding.
Acinetobacter baumannii
is a human bacterial pathogen of increasing concern in clinical settings due to the emergence of antibiotic resistant strains and the lack of effective therapeutics. ...Researchers have been exploring new treatment options such as novel drug candidates and vaccines to prevent severe infections and mortality. Bacterial surface antigens that are essential to
A. baumannii
for acquiring micronutrients (e.g. iron, zinc) from nutrient restricted environments are being considered as targets for vaccines or immunotherapy due to their crucial role for growth and pathogenesis in the human host. BauA, the outer membrane receptor for the siderophore acinetobactin was targeted for vaccine development in this study. Due to challenges in the commercial production of membrane proteins for vaccines, a novel hybrid antigen method developed by our group was used. Exposed loops of BauA were selected and displayed on a foreign scaffold to generate novel hybrid antigens designed to elicit an immune response against the native BauA protein. The potential epitopes were incorporated into a scaffold derived from the C-lobe of
Neisseria meningitidis
transferrin binding protein B (TbpB), named the loopless C-lobe (LCL). Hybrid proteins displaying three selected loops (5, 7 and 8) individually or in combination were designed and produced and evaluated in an
A. baumannii
murine sepsis model as vaccine antigens. Immunization with the recombinant BauA protein protected 100% of the mice while immunization with hybrid antigens displaying individual loops achieved between 50 and 100% protection. The LCL scaffold did not induce a protective immune response, enabling us to attribute the observed protection elicited by the hybrid antigens to the displayed loops. Notably, the mice immunized with the hybrid antigen displaying loop 7 were completely protected from infection. Taken together, these results suggest that our hybrid antigen approach is a viable method for generating novel vaccine antigens that target membrane surface proteins necessary for bacterial growth and pathogenesis and the loop 7 hybrid antigen can be a foundation for approaches to combat
A. baumannii
infections.
Bovine respiratory disease complex (BRDc) is a major cause of morbidity and mortality in beef cattle. There is recent evidence suggesting that the nasopharyngeal microbiota has a key role in ...respiratory health and disease susceptibility in cattle. However, there is a paucity of knowledge regarding evolution of the nasopharyngeal microbiota when cattle are most likely to develop BRDc (i.e., from weaning to 40days after arrival at a feedlot). The objective was to describe the evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40days after arrival at a feedlot. Deep nasal swabs (DNS) from 30 Angus-cross steers were collected at weaning, on arrival at a feedlot, and at day 40 after arrival. The DNA was extracted from DNS and the hypervariable region V3 of the 16S rRNA gene was amplified and sequenced (Illumina MiSeq platform). Nasopharyngeal microbiota underwent a profound evolution from weaning to arrival at the feedlot and from arrival to day 40, with the abundance of 92 Operational Taxonomic Units (OTUs) significantly changing over time. Mycoplasma (M. dispar and M. bovirhinis) was the most abundant genus in the nasopharynx, accounting for 53% of the total bacterial population. Because an evolving bacterial community may be less capable of resisting colonization by pathogenic bacteria, the instability of the nasopharyngeal microbiota documented in this study might explain why cattle are most likely to be affected with BRDc during the first weeks after weaning and arrival at a feedlot.
•Acinetobacter baumannii infections are difficult-to-treat with high mortality.•The immunogenic loops from BauA and Omp34 were used as a hybrid antigen.•C-lobe of TbpB surface lipoprotein was used to ...display BauA and Omp34 loops.•The combination of both loops brought about 71.43% protection against A. baumannii.•Multivalent vaccine induces broadly reactive antibody responses against A. baumannii.
The complexity of treating Acinetobacter baumannii infections with the newly developed resistant strains has led researchers to confront this pathogen by developing vaccines. In this study, we used two important virulence factors of A. baumannii to elicit immunity against the A. baumannii. The immunogenic loops were from Baumannii acinetobactin utilization A (BauA) and 34kD outer membrane protein (Omp34). C-lobe derivative of the TbpB surface lipoprotein was used to display the superficial epitopes of the TbpA receptor protein of Neisseria meningitidis. The resulting loopless C-lobe (LCL) with implanted nucleotide sequences of the immunogenic loops from BauA and Omp34 was used as a hybrid antigen. The hybrid antigens were expressed in the E. coli and were used to immunize mice. The mice were challenged with a clinical isolate of A. baumannii (ABI022). Immunization with the hybrid antigens of the BauA loop 7 (BauAL7P3), Omp34 loop 3 Omp34L3P1, and the combination of both loops (BauAL7P3Omp34L3P1) brought about 42.86%, 42.86%, and 71.43% protection against A. baumannii infection. Histopathological findings in the immunized mice showed bronchioles clear from inflammatory cells and normal texture of the spleen and liver. The findings support the use of a multivalent vaccine to induce broadly reactive antibody responses against heterologous A. baumannii strains.
Structure-based approaches to the delineation of immunogens for vaccine development have a throughput requirement that is difficult to meet in practice with conventional methods of structure ...determination. Here we present a strategy for rapid and accurate structure generation in support of antigen engineering programs. The approach is developed around the modeling of interactions between host transferrin (Tf) and the bacterial vaccine target transferrin binding protein B (TbpB) from Gram-negative pathogens such as Neisseria meningitidis. Using an approach based solely on cross-linking mass spectrometry (XL-MS) data, monomeric structural models, and the Integrative Modeling Platform (IMP), we demonstrate that converged representations of the Tf:TbpB interactions can be returned that accurately reflect the binding interface and the relative orientation of the monomeric units, with the capacity to scale to the analysis of interactions from any number of additional strains. We show that a key element to accurate modeling involves the application of hetero-bifunctional cross-linkers incorporating fast-acting photoactivatable diazirines coupled with conventional amine-targeting N-hydroxysuccinimide esters, and we demonstrate that conventional homo-bifunctional reagents used in cross-linking kinetically trap dynamic states in the ensemble. Therefore, the application of both classes of cross-linker provides an opportunity to empirically detect protein dynamics during integrative structural modeling.
is an important human pathogen causing substantial mortality in hospitalized patients for which treatment with antibiotics has become problematic due to growing antibiotic resistance. In an attempt ...to develop alternative strategies for dealing with these serious infections surface antigens are being considered as targets for vaccines or immunotherapy. The surface receptor proteins required for zinc acquisition in Gram-negative bacterial pathogens have been proposed as vaccine targets due to their crucial role for growth in the human host. In this study we selected the putative ZnuD outer membrane receptor from
as a target for vaccine development. Due to challenges in production of an integral outer membrane protein for vaccine production, we adopted a recently described hybrid antigen approach in which surface epitopes from the
TbpA receptor protein were displayed on a derivative of the C-lobe of the surface lipoprotein TbpB, named the loopless C-lobe (LCL). A structural model for ZnuD was generated and four surface loops were selected for hybrid antigen production by computational approaches. Hybrid antigens were designed displaying the four selected loops (2, 5, 7, and 11) individually or together in a single hybrid antigen. The hybrid antigens along with ZnuD and the LCL scaffold were produced in the
cytoplasm either as soluble antigens or as inclusion bodies, that were used to generate soluble antigens upon refolding. Mice were immunized with the hybrid antigens, ZnuD or LCL and then used in an
sepsis model to evaluate their ability to protect against infection. As expected, the LCL scaffold did not induce a protective immune response, enabling us to attribute observed protection to the displayed loops. Immunization with the refolded ZnuD protein protected 63% of the mice while immunization with hybrid antigens displaying individual loops achieved between 25 and 50% protection. Notably, the mice immunized with the hybrid antigen displaying the four loops were completely protected from infection.
While developing vaccines targeting surface transferrin receptor proteins in Gram-negative pathogens of humans and food production animals, the common features derived from their evolutionary origins ...has provided us with insights on how improvements could be implemented in the various stages of research and vaccine development. These pathogens are adapted to live exclusively on the mucosal surfaces of the upper respiratory or genitourinary tract of their host and rely on their receptors to acquire iron from transferrin for survival, indicating that there likely are common mechanisms for delivering transferrin to the mucosal surfaces that should be explored. The modern-day receptors are derived from those present in bacteria that lived over 320 million years ago. The pathogens represent the most host adapted members of their bacterial lineages and may possess factors that enable them to have strong association with the mucosal epithelial cells, thus likely reside in a different niche than the commensal members of the bacterial lineage. The bacterial pathogens normally lead a commensal lifestyle which presents challenges for development of relevant infection models as most infection models either exclude the early stages of colonization or subsequent disease development, and the immune mechanisms at the mucosal surface that would prevent disease are not evident. Development of infection models emulating natural horizontal disease transmission are also lacking. Our aim is to share our insights from the study of pathogens of humans and food production animals with individuals involved in vaccine development, maintaining health or regulation of products in the human and animal health sectors.
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