Paul Adamiak

Bacterial receptors for host transferrin and lactoferrin: molecular mechanisms and role in host–microbe interactions

Iron homeostasis in the mammalian host limits the availability of iron to invading pathogens and is thought to restrict iron availability for microbes inhabiting mucosal surfaces. The presence of surface receptors for the host iron-binding glycoproteins transferrin (Tf) and lactoferrin (Lf) in globally important Gram-negative bacterial pathogens of humans and food production animals suggests that Tf and Lf are important sources of iron in the upper respiratory or genitourinary tracts, where they exclusively reside. Lf receptors have the additional function of protecting against host cationic antimicrobial peptides, suggesting that the bacteria expressing these receptors reside in a niche where exposure is likely. In this review we compare Tf and Lf receptors with respect to their structural and functional features, their role in colonization and infection, and their distribution among pathogenic and commensal bacteria.

The role of lactoferrin binding protein B in mediating protection against human lactoferricin

Bacteria that inhabit the mucosal surfaces of the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment because of iron sequestration by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic, antimicrobial peptide lactoferricin is produced by proteolysis of lactoferrin. Several Gram-negative pathogens express a lactoferrin receptor that enables the bacteria to use lactoferrin as an iron source. The receptor is composed of an integral membrane protein, lactoferrin binding protein A (LbpA), and a membrane-bound lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for growth with lactoferrin as the sole iron source, whereas the role of LbpB in iron acquisition is not yet known. In this study, we demonstrate that LbpB from 2 different species is capable of providing protection against the killing activity of a human lactoferrin-derived peptide. We investigated the prevalence of lactoferrin receptors in bacteria and examined their sequence diversity. We propose that the protection against the cationic antimicrobial human lactoferrin-derived peptide is associated with clusters of negatively charged amino acids in the C-terminal lobe of LbpB that is a common feature of this protein.

Patterns of sequence variation within the Neisseria meningitidis lactoferrin binding proteins.

Lactoferrin binding proteins A and B (LbpA and LbpB) compose the lactoferrin receptor of the obligate human pathogen Neisseria meningitidis . This receptor is thought to be important for colonization and initiation of invasive disease because of its role in acquiring host iron and providing protection from the cationic peptide, lactoferricin. By virtue of its function, the receptor is accessible to the host immune system and displays substantial sequence variation. In this study, we analyzed a broad collection of LbpAs (62) and LbpBs (101) to determine the distribution of sequence variation within each protein and to search for patterns between sequence similarity and strain typing. The sequence variation in LbpA was predominantly observed in 3 surface loops and, surprisingly, in the N-terminal region immediately upstream of the predicted TonB box. The analysis of LbpB revealed that the variability was distributed throughout the protein, particularly in the highly variable negatively charged regions in the C-lobe, but otherwise was greater in the N-lobe than the C-lobe. There was no readily identifiable correlation between the sequence variation within LbpA, LbpB, multi-locus sequence type, or serogroup.

Sequence and structural diversity of transferrin receptors in Gram-negative porcine pathogens.

Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis are bacterial pathogens from the upper respiratory tract that are responsible for a substantial burden of porcine disease. Although reduction of disease has been accomplished by intensive management practices, immunization remains an important strategy for disease prevention, particularly when intensive management practices are not feasible or suitable. An attractive target for vaccine development is the surface receptor involved in acquiring iron from host transferrin, since it is common to all three pathogenic species and has been shown to be essential for survival and disease causation. It has also recently been demonstrated that an engineered antigen derived from the lipoprotein component of the receptor, transferrin-binding protein B (TbpB), was more effective at preventing infection by H. parasuis than a commercial vaccine product. This study was initiated to explore the genetic and immunogenic diversity of the transferrin receptor system from these species. Nucleic acid sequences were obtained from a geographically and temporally diverse collection of isolates, consisting of 41 A. pleuropneumoniae strains, 30 H. parasuis strains, and 2 A. suis strains. Phylogenetic analyses demonstrated that the receptor protein sequences cluster independently of species, suggesting that there is genetic exchange between these species such that receptor-based vaccines should logically target all three species. To evaluate the cross-reactive response of TbpB-derived antigens, pigs were immunized with the intact TbpB, the TbpB N-lobe and the TbpB C-lobe from A. pleuropneumoniae strain H49 and the resulting sera were tested against a representative panel of TbpBs; demonstrating that the C-lobe induces a broadly cross-reactive response. Overall our results indicate that there is a common reservoir for transferrin receptor antigenic variation amongst these pathogens. While this could present a challenge to future vaccine development, our results suggest a rationally designed TbpB-based vaccine may provide protection against all three pathogens.

Patterns of structural and sequence variation within isotype lineages of the Neisseria meningitidis transferrin receptor system.

Neisseria meningitidis inhabits the human upper respiratory tract and is an important cause of sepsis and meningitis. A surface receptor comprised of transferrin‐binding proteins A and B (TbpA and TbpB), is responsible for acquiring iron from host transferrin. Sequence and immunological diversity divides TbpBs into two distinct lineages; isotype I and isotype II. Two representative isotype I and II strains, B16B6 and M982, differ in their dependence on TbpB for in vitro growth on exogenous transferrin. The crystal structure of TbpB and a structural model for TbpA from the representative isotype I N. meningitidis strain B16B6 were obtained. The structures were integrated with a comprehensive analysis of the sequence diversity of these proteins to probe for potential functional differences. A distinct isotype I TbpA was identified that co‐varied with TbpB and lacked sequence in the region for the loop 3 α‐helix that is proposed to be involved in iron removal from transferrin. The tightly associated isotype I TbpBs had a distinct anchor peptide region, a distinct, smaller linker region between the lobes and lacked the large loops in the isotype II C‐lobe. Sequences of the intact TbpB, the TbpB N‐lobe, the TbpB C‐lobe, and TbpA were subjected to phylogenetic analyses. The phylogenetic clustering of TbpA and the TbpB C‐lobe were similar with two main branches comprising the isotype 1 and isotype 2 TbpBs, possibly suggesting an association between TbpA and the TbpB C‐lobe. The intact TbpB and TbpB N‐lobe had 4 main branches, one consisting of the isotype 1 TbpBs. One isotype 2 TbpB cluster appeared to consist of isotype 1 N‐lobe sequences and isotype 2 C‐lobe sequences, indicating the swapping of N‐lobes and C‐lobes. Our findings should inform future studies on the interaction between TbpB and TbpA and the process of iron acquisition.

The specificity of protection against cationic antimicrobial peptides by lactoferrin binding protein B

A variety of Gram-negative pathogens possess host-specific lactoferrin (Lf) receptors that mediate the acquisition of iron from host Lf. The integral membrane protein component of the receptor, lactoferrin binding protein A specifically binds host Lf and is required for acquisition of iron from Lf. In contrast, the role of the bi-lobed surface lipoprotein, lactoferrin binding protein B (LbpB), in Lf binding and iron acquisition is uncertain. A common feature of LbpBs from most species is the presence of clusters of negatively charged amino acids in the protein’s C-terminal lobe. Recently it has been shown that the negatively charged regions from the Neisseria meningitidis LbpB are responsible for protecting against an 11 amino acid cationic antimicrobial peptide (CAP), lactoferricin (Lfcin), derived from human Lf. In this study we investigated whether the LbpB confers resistance to other CAPs since N. meningitidis is likely to encounter other CAPs from the host. LbpB provided protection against the cathelicidin derived peptide, cathelicidin related antimicrobial peptide (mCRAMP), but did not confer protection against Tritrp 1 or LL37 under our experimental conditions. When tested against a range of rationally designed synthetic peptides, LbpB was shown to protect against IDR-1002 and IDR-0018 but not against HH-2 or HHC10.

Beyond bricks and mortar: a rural network approach to preclinical medical education

BackgroundCountries with expansive rural regions often experience an unequal distribution of physicians between rural and urban communities. A growing body of evidence suggests that the exposure to positive rural learning experiences has an influence on a physician’s choice of practice location. Capitalizing on this observation, many medical schools have developed approaches that integrate rural exposure into their curricula during clerkship. It is postulated that a preclinical rural exposure may also be effective. However, to proceed further in development, accreditation requirements must be considered. In this investigation, academic equivalence between a preclinical rural community based teaching method and the established education model was assessed.MethodTwo separate preclinical courses from the University of Calgary’s three year Undergraduate Medical program were taught at two different rural sites in 2010 (11 students) and 2012 (12 students). The same academic content was delivered in the pilot sites as in the main teaching centre. To ensure consistency of teaching skills, faculty development was provided at each pilot site. Academic equivalence between the rural based learners and a matched cohort at the main University of Calgary site was determined using course examination scores, and the quality of the experience was evaluated through learner feedback.ResultsIn both pilot courses there was no significant difference between examination scores of the rural distributed learners and the learners at the main University of Calgary site (p > 0.05). Feedback from the participating students demonstrated that the preceptors were very positively rated and, relative to the main site, the small group learning environment appeared to provide strengthened social support.ConclusionThese results suggest that community distributed education in pre-clerkship may offer academically equivalent training to existing traditional medical school curricula while also providing learners with positive rural social learning environments. The approach described may offer the potential to increase exposure to rural practice without the cost of constructing additional physical learning sites.

Specialty resident perceptions of the impact of a distributed education model on practice location intentions

Abstract Objectives: There is an increased focus internationally on the social mandate of postgraduate training programs. This study explores specialty residents’ perceptions of the impact of the University of Calgary’s (UC) distributed education rotations on their self-perceived likelihood of practice location, and if this effect is influenced by resident specialty or stage of program. Methods: Residents participating in the UC Distributed Royal College Initiative (DistRCI) between July 2010 and June 2013 completed an online survey following their rotation. Descriptive statistics and student’s t-test were employed to analyze quantitative survey data, and a constant comparative approach was used to analyze free text qualitative responses. Results: Residents indicated they were satisfied with the program (92%), and that the distributed rotations significantly increased their self-reported likelihood of practicing in smaller centers (p < 0.05). The findings suggest that the shift in attitude is independent of discipline, program year, and logistical experiences of living at the distributed sites, and is consistent across multiple cohorts over several academic years. Conclusion: The findings highlight the value of a distributed education program in contributing to future practice and career development, and its relevance in the social accountability of postgraduate programs.