The heparin-binding hemagglutinin protein of Mycobacterium tuberculosis is a nucleoid-associated protein.

Nucleoid-associated proteins (NAPs) regulate multiple cellular processes such as gene expression, virulence, and dormancy throughout bacterial species. NAPs help in the survival and adaptation of Mycobacterium tuberculosis (Mtb) within the host. Fourteen NAPs have been identified in Escherichia coli; however, only seven NAPs are documented in Mtb. Given its complex lifestyle, it is reasonable to assume that Mtb would encode for more NAPs. Using bioinformatics tools and biochemical experiments, we have identified the heparin-binding hemagglutinin (HbhA) protein of Mtb as a novel sequence-independent DNA-binding protein which has previously been characterized as an adhesion molecule required for extrapulmonary dissemination. Deleting the carboxy-terminal domain of HbhA resulted in a complete loss of its DNA-binding activity. Atomic force microscopy showed HbhA-mediated architectural modulations in the DNA, which may play a regulatory role in transcription and genome organization. Our results showed that HbhA colocalizes with the nucleoid region of Mtb. Transcriptomics analyses of a hbhA KO strain revealed that it regulates the expression of ∼36% of total and ∼29% of essential genes. Deletion of hbhA resulted in the upregulation of ∼73% of all differentially expressed genes, belonging to multiple pathways suggesting it to be a global repressor. The results show that HbhA is a nonessential NAP regulating gene expression globally and acting as a plausible transcriptional repressor.

Manufacture of a Stable Lyophilized Formulation of the Live Attenuated Pertussis Vaccine BPZE1.

Current pertussis vaccines protect against disease, but not against colonization by and transmission of Bordetella pertussis, whereas natural infection protects against both. The live attenuated vaccine BPZE1 was developed to mimic immunogenicity of natural infection without causing disease, and in preclinical models protected against pertussis disease and B. pertussis colonization after a single nasal administration. Phase 1 clinical studies showed that BPZE1 is safe and immunogenic in humans when administered as a liquid formulation, stored at ≤-70 °C. Although BPZE1 is stable for two years at ≤-70 °C, a lyophilized formulation stored at ≥5 °C is required for commercialization. The development of a BPZE1 drug product, filled and lyophilized directly in vials, showed that post-lyophilization survival of BPZE1 depended on the time of harvest, the lyophilization buffer, the time between harvest and lyophilization, as well as the lyophilization cycle. The animal component-free process, well defined in terms of harvest, processing and lyophilization, resulted in approximately 20% survival post-lyophilization. The resulting lyophilized drug product was stable for at least two years at -20 °C ± 10 °C, 5 °C ± 3 °C and 22.5 °C ± 2.5 °C and maintained its vaccine potency, as evaluated in a murine protection assay. This manufacturing process thus enables further clinical and commercial development of BPZE1.

Development and Standardization of a High-Throughput Bordetella pertussis Growth-Inhibition Assay.

Bordetella pertussis, the main causative agent of whooping cough, is a reemerging pathogen, and recent vaccine-resistant strain outbreaks and emergence of macrolides-resistant strains in China raised new concerns for control of the disease. New vaccines and potentially new antibiotics are thus needed. B. pertussis is tedious to culture and requires several days of growth to count isolated colonies on agar-based media, making large-scale screening of new anti-B. pertussis compounds or functional evaluation of large sample sizes of immune sera difficult. Here, we developed a scalable, rapid, high-throughput luminescence-based Bordetella growth inhibition assay (BGIA) to quantify surviving bacteria after treatment with anti-B. pertussis compounds. A strong correlation between luminescence and colony-forming units (r2 = 0.9345, p < 0.0001) was found and the BGIA showed high sensitivity and reproducibility. We demonstrate here that the BGIA can be used to quantify resistance of B. pertussis to antibiotics, sensitivity to complement and to human serum in an easy-to-operate and fast manner. We have optimized the assay and tested the effects of different B. pertussis strains and growth conditions on serum and complement sensitivity. We also uncovered complement-independent antibody-mediated inhibition of B. pertussis growth. The BGIA can thus effectively be implemented for large-scale serum studies to further investigate anti-B. pertussis immune responses at a functional level, as well as for screening of B. pertussis strains for their resistance to antibiotics or complement, and for high-throughput screening of novel anti-B. pertussis compounds.

Quantity and Quality of Antibodies After Acellular Versus Whole-cell Pertussis Vaccines in Infants Born to Mothers Who Received Tetanus, Diphtheria, and Acellular Pertussis Vaccine During Pregnancy: A Randomized Trial.

BACKGROUND: The blunting effect of pertussis immunization during pregnancy on infant antibody responses induced by whole-cell pertussis (wP) vaccination is not well-defined. METHODS: This randomized controlled trial (NCT02408926) followed term infants born to mothers vaccinated with tetanus, diphtheria, and acellular pertussis (Tdap) vaccine during pregnancy in Thailand. Infants received either acellular pertussis (aP)- or wP-containing vaccine at 2, 4, 6, and 18 months of age. A comparison group comprised wP-vaccinated children born to mothers not vaccinated during pregnancy. Antibodies against pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (PRN) were evaluated using commercial enzyme-linked immunosorbent assays. Functionality of antibodies against Bordetella pertussis was measured using Bordetella pertussis growth inhibition assay. RESULTS: After maternal Tdap vaccination, 158 infants vaccinated with aP-containing vaccines possessed higher antibody levels (P < .001) against all tested B. pertussis antigens postpriming compared to 157 infants receiving wP-containing vaccines. At 1 month postbooster, only anti-FHA and anti-PRN antibodies were still significantly higher (P < .001) in the aP group. Significantly higher anti-PT and anti-FHA (P < .001), but not anti-PRN immunoglobulin G, were observed among 69 wP-vaccinated infants born to control mothers compared with wP-vaccinated infants of Tdap-vaccinated mothers after primary and booster vaccination. The antibody functionality was higher in all wP-vaccinated infants at all times. CONCLUSIONS: Maternal Tdap vaccination inhibited more pertussis-specific responses in wP-vaccinated infants compared to aP-vaccinated infants, and the control group of unvaccinated women had highest PT-specific responses, persisting until after the booster dose. Antibody functionality was better in the wP groups. CLINICAL TRIALS REGISTRATION: NCT02408926.Infant whole-cell pertussis (wP) vaccine responses are blunted after maternal Tdap vaccination. Pertussis antibody titers are higher in acellular pertussis (aP)- than wP-vaccinated infants of immunized mothers, yet quality of antibodies, measured as serum-mediated bacterial growth inhibition, is better after wP than aP vaccination.

Coordinate regulation of virulence and metabolic genes by the transcription factor HbhR in Mycobacterium marinum.

The heparin-binding hemagglutinin (HBHA) is a multifunctional protein involved in adherence of Mycobacterium tuberculosis to non-phagocytic cells and in the formation of intracytosolic lipid inclusions. We demonstrate that the expression of hbhA is regulated by a transcriptional repressor, named HbhR, in Mycobacterium marinum. The hbhR gene, located upstream of hbhA, was identified by screening a transposon insertion library and detailed analysis of a mutant overproducing HBHA. HbhR was found to repress both hbhA and hbhR transcription by binding to the promoter regions of both genes. Complementation restored production of HBHA. RNA-seq analyses comparing the mutant and parental strains uncovered 27 genes, including hbhA, that were repressed and 20 genes activated by HbhR. Among the former, the entire locus of genes coding for a type-VII secretion system, including esxA, esxB and pe-ppe paralogs, as well as the gene coding for PspA, present in intracellular lipid vesicles, was identified, as was katG, a gene involved in the sensitivity to isoniazid. The latter category contains genes that play a role in diverse functions, such as metabolism and resistance to oxidative conditions. Thus, HbhR appears to be a master regulator, linking the transcriptional regulation of virulence, metabolic and antibiotic sensitivity genes in M. marinum.

Heparin-Binding Hemagglutinin Adhesin (HBHA) Is Involved in Intracytosolic Lipid Inclusions Formation in Mycobacteria.

The heparin-binding hemagglutinin adhesin (HBHA) is an important virulence factor of Mycobacterium tuberculosis. It is a surface-displayed protein that serves as an adhesin for non-phagocytic cells and is involved in extra-pulmonary dissemination of the tubercle bacillus. It is also an important latency antigen useful for the diagnosis of latently M. tuberculosis-infected individuals. Using fluorescence time-lapse microscopy on mycobacteria that produce HBHA-green fluorescent protein chimera, we show here that HBHA can be found at two different locations and dynamically alternates between the mycobacterial surface and the interior of the cell, where it participates in the formation of intracytosolic lipid inclusions (ILI). Compared to HBHA-producing mycobacteria, HBHA-deficient mutants contain significantly lower amounts of ILI when grown in vitro or within macrophages, and the sizes of their ILI are significantly smaller. Lipid-binding assays indicate that HBHA is able to specifically bind to phosphatidylinositol and in particular to 4,5 di-phosphorylated phosphatidylinositol, but not to neutral lipids, the main constituents of ILI. HBHA derivatives lacking the C-terminal methylated, lysine-rich repeat region fail to bind to these lipids and these derivatives also fail to complement the phenotype of HBHA-deficient mutants. These studies indicate that HBHA is a moonlighting protein that serves several functions depending on its location. When surface exposed, HBHA serves as an adhesin, and when intracellularly localized, it participates in the generation of ILI, possibly as a cargo to transport phospholipids from the plasma membrane to the ILI in the process of being formed.

Rv0613c/MSMEG_1285 Interacts with HBHA and Mediates Its Proper Cell-Surface Exposure in Mycobacteria.

Heparin-binding haemagglutinin (HBHA) is a surface-exposed virulence factor of Mycobacterium tuberculosis and is involved in the binding of mycobacteria to non-phagocytic cells, allowing for extra-pulmonary dissemination of the bacilli. Despite its surface exposure, HBHA is not produced as a pre-protein containing a typical cleavable N-terminal signal peptide and is thus likely secreted by a Sec-independent, as of yet unknown mechanism. Here, we used the bacterial adenylate cyclase two-hybrid system to identify the proteins encoded by rv0613c and mmpL14 as being able to interact with HBHA. Our study was focused on Rv0613c, as it showed more consistent interactions with HBHA than MmpL14. Deletion of its orthologous gene MSMEG_1285 in recombinant Mycobacterium smegmatis producing HBHA from M. tuberculosis resulted in the loss of proper surface exposure of HBHA, as evidenced by atomic force microscopy. Furthermore, the lack of MSMEG_1285 also abolished the clumping phenotype and rough colony morphology of the recombinant M. smegmatis and reduced its adherence to A549 epithelial cells. These phenotypes have previously been associated with surface-exposed HBHA. Thus, MSMEG_1285 is directly involved in the proper cell-surface exposure of HBHA. These observations identify MSMEG_1285/Rv0613c as the first accessory protein involved in the cell surface exposure of HBHA.

Construction and evaluation of Bordetella pertussis live attenuated vaccine strain BPZE1 producing Fim3.

Pertussis or whooping cough is currently the most prevalent vaccine-preventable childhood disease despite >85% global vaccination coverage. In recent years incidence has greatly increased in several high-income countries that have switched from the first-generation, whole-cell vaccine to the newer acellular vaccines, calling for improved vaccination strategies with better vaccines. We have developed a live attenuated pertussis vaccine candidate, called BPZE1, which is currently in clinical development. Unlike other pertussis vaccines, BPZE1 has been shown to provide strong protection against infection by the causative agent of pertussis, Bordetella pertussis, in non-human primates. BPZE1 is a derivative of the B. pertussis strain Tohama I, which produces serotype 2 (Fim2) but not serotype 3 fimbriae (Fim3). As immune responses to fimbriae are likely to contribute to protection, we constructed a BPZE1 derivative, called BPZE1f3, that produces both serotypes of fimbriae. Whereas nasal vaccination of mice with BPZE1 induced antibodies to Fim2 but not to Fim3, vaccination with BPZE1f3 elicited antibodies to both Fim2 and Fim3 at approximately the same level. In mice, both BPZE1 and BPZE1f3 provided equal levels of protection against clinical isolates that either produce Fim2 alone, both Fim2 and Fim3, or no fimbriae. However, vaccination with BPZE1f3 provided significantly stronger protection against Fim3-only producing B. pertussis than vaccination with BPZE1, indicating that immune responses to fimbriae contribute to serotype-specific protection against B. pertussis infection.

Diversion of complement-mediated killing by Bordetella.

The complement cascade participates in protection against bacterial infections, and pathogens, including Bordetella pertussis, have developed complement-evading strategies. Here we discuss current knowledge on B. pertussis complement evasion strategies and the role of antibody-dependent complement-mediated killing in protection against B. pertussis infection pointing out important knowledge gaps for further research to improve current pertussis vaccines.

HBHA vaccination may require both Th1 and Th17 immune responses to protect mice against tuberculosis.

Almost one century after the discovery of the BCG vaccine, tuberculosis remains a major cause of global mortality and morbidity, emphasizing the urgent need to design more efficient vaccines. The heparin-binding haemagglutinin (HBHA) appears to be a promising vaccine candidate, as it was shown to afford protection to mice against a challenge infection with Mycobacterium tuberculosis when combined with the strong adjuvant DDA/MPL (dimethyldioctadecyl-ammonium bromide/monophosphoryl lipid A), a TLR4 ligand. In this study, we investigated the immunological response and protection of mice immunized with HBHA formulated in lipid-containing nanoparticles and adjuvanted with CpG, a TLR9 ligand. Subcutaneous immunization with this HBHA formulation led to a marked Th1 response, characterized by high IFN-γ levels, but no significant IL-17 production, both in spleen and lung, in contrast to DDA/MPL MPL-formulated HBHA, which induced both IFN-γ and IL-17. This cytokine profile was also observed in BCG-primed mice and persisted after M. tuberculosis infection. No significant protection was obtained against challenge infection after vaccination with the nanoparticle-CpG formulation, and this was associated with a failure to mount a memory immune response. These results suggest the importance of both Th1 and Th17 immune responses for vaccine-induced immunity.

Attenuated Bordetella pertussis vaccine protects against respiratory syncytial virus disease via an IL-17-dependent mechanism.

RATIONALE: We attenuated virulent Bordetella pertussis by genetically eliminating or detoxifying three major toxins. This strain, named BPZE1, is being developed as a possible live nasal vaccine for the prevention of whooping cough. It is immunogenic and safe when given intranasally in adult volunteers. OBJECTIVES: Before testing in human infants, we wished to examine the potential effect of BPZE1 on a common pediatric infection (respiratory syncytial virus [RSV]) in a preclinical model. METHODS: BPZE1 was administered before or after RSV administration in adult or neonatal mice. Pathogen replication, inflammation, immune cell recruitment, and cytokine responses were measured. MEASUREMENTS AND MAIN RESULTS: BPZE1 alone did not cause overt disease, but induced efflux of neutrophils into the airway lumen and production of IL-10 and IL-17 by mucosal CD4(+) T cells. Given intranasally before RSV infection, BPZE1 markedly attenuated RSV, preventing weight loss, reducing viral load, and attenuating lung cell recruitment. Given neonatally, BPZE1 also protected against RSV-induced weight loss even through to adulthood. Furthermore, it markedly increased IL-17 production by CD4(+) T cells and natural killer cells and recruited regulatory cells and neutrophils after virus challenge. Administration of anti-IL-17 antibodies ablated the protective effect of BPZE1 on RSV disease. CONCLUSIONS: Rather than enhancing RSV disease, BPZE1 protected against viral infection, modified viral responses, and enhanced natural mucosal resistance. Prevention of RSV infection by BPZE1 seems in part to be caused by induction of IL-17. Clinical trial registered with www.clinicaltrials.gov (NCT 01188512).

Novel feature of Mycobacterium avium subsp. paratuberculosis, highlighted by characterization of the heparin-binding hemagglutinin adhesin.

Mycobacterium avium subsp. paratuberculosis comprises two genotypically defined groups, known as the cattle (C) and sheep (S) groups. Recent studies have reported phenotypic differences between M. avium subsp. paratuberculosis groups C and S, including growth rates, infectivity for macrophages, and iron metabolism. In this study, we investigated the genotypes and biological properties of the virulence factor heparin-binding hemagglutinin adhesin (HBHA) for both groups. In Mycobacterium tuberculosis, HBHA is a major adhesin involved in mycobacterium-host interactions and extrapulmonary dissemination of infection. To investigate HBHA in M. avium subsp. paratuberculosis, we studied hbhA polymorphisms by fragment analysis using the GeneMapper technology across a large collection of isolates genotyped by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP-IS900) analyses. Furthermore, we analyzed the structure-function relationships of recombinant HBHA proteins of types C and S by heparin-Sepharose chromatography and surface plasmon resonance (SPR) analyses. In silico analysis revealed two forms of HBHA, corresponding to the prototype genomes for the C and S types of M. avium subsp. paratuberculosis. This observation was confirmed using GeneMapper on 85 M. avium subsp. paratuberculosis strains, including 67 strains of type C and 18 strains of type S. We found that HBHAs from all type C strains contain a short C-terminal domain, while those of type S present a long C-terminal domain, similar to that produced by Mycobacterium avium subsp. avium. The purification of recombinant HBHA from M. avium subsp. paratuberculosis of both types by heparin-Sepharose chromatography highlighted a correlation between their affinities for heparin and the lengths of their C-terminal domains, which was confirmed by SPR analysis. Thus, types C and S of M. avium subsp. paratuberculosis may be distinguished by the types of HBHA they produce, which differ in size and adherence properties, thereby providing new evidence that strengthens the genotypic differences between the C and S types of M. avium subsp. paratuberculosis.

Immunogenicity of live attenuated B. pertussis BPZE1 producing the universal influenza vaccine candidate M2e.

BACKGROUND: Intranasal delivery of vaccines directed against respiratory pathogens is an attractive alternative to parenteral administration. However, using this delivery route for inactivated vaccines usually requires the use of potent mucosal adjuvants, and no such adjuvant has yet been approved for human use. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a live attenuated Bordetella pertussis vaccine, called BPZE1, and show here that it can be used to present the universal influenza virus epitope M2e to the mouse respiratory tract to prime for protective immunity against viral challenge. Three copies of M2e were genetically fused to the N-terminal domain of filamentous hemagglutinin (FHA) and produced in recombinant BPZE1 derivatives in the presence or absence of endogenous full-length FHA. Only in the absence of FHA intranasal administration of the recombinant BPZE1 derivative induced antibody responses to M2e and effectively primed BALB/c mice for protection against influenza virus-induced mortality and reduced the viral load after challenge. Strong M2e-specific antibody responses and protection were observed after a single nasal administration with the recombinant BPZE1 derivative, followed by a single administration of M2e linked to a virus-like particle without adjuvant, whereas priming alone with the vaccine strain did not protect. CONCLUSIONS/SIGNIFICANCE: Using recombinant FHA-3M2e-producing BPZE1 derivatives for priming and the universal influenza M2e peptide linked to virus-like particles for boosting may constitute a promising approach for needle-free and adjuvant-free nasal vaccination against influenza.

Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis.

BACKGROUND: Paratuberculosis remains today a major global problem in animal health, especially for dairy cattle. However, the diagnosis of its etiologic agent, Mycobacterium avium subsp. paratuberculosis (Map), still lacks sensitivity because of the lack of available antigens. Little is known about the virulence factors for this pathogen. In this study we have developed a method to produce and purify the heparin-binding hemagglutinin (HBHA), a major adhesin of Mycobacteria, from a culture of Map. FINDINGS: For this extremely slow-growing Mycobacterium, a culture was established in a 3-liter bioreactor. Using the bioreactor the amount of the Map biomass was increased 5-fold compared to a classical culture in flasks. The map-HBHA was purified from a Map lysate by heparin-Sepharose chromatography on HiTrap columns. Binding of map-HBHA onto heparin-Sepharose can be reduced in the presence of salt. Consequently, all steps of sample preparation and column equilibration were carried out in 20 mM Tris-HCl (pH 7.2). The map-HBHA was eluted by a linear NaCl gradient. High resolution mass spectrometry analyses revealed that the native form of map-HBHA has posttranslational modifications, including the removal of the initiation methionine, acetylation of the alanine residue at the N-terminal extremity and the presence of methylated lysines in the C-terminal domain of the protein. CONCLUSIONS: An optimized culture of Map in a bioreactor was established to purify the native map-HBHA from a Map lysate by heparin-Sepharose chromatography. The availability of this antigen offers the possibility to study the structure of the protein and to examine its role in pathogenicity, in particular to better understand the specific interactions of Map with the intestinal tissue. The map-HBHA obtained in its native immunogenic form may also be useful to improve the diagnostic test, especially for the development of a new T-cell-based interferon gamma release assays.

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence

When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

Dual mechanism of protection by live attenuated Bordetella pertussis BPZE1 against Bordetella bronchiseptica in mice.

Bordetella bronchiseptica, a gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including man, and no human vaccine is currently available. Acellular pertussis vaccines protect poorly against B. bronchiseptica, although they contain cross-reactive antigens. We have recently developed Bordetella pertussis BPZE1, a novel, live attenuated pertussis vaccine, currently completing phase I clinical trials in humans, and found that it protects against both B. pertussis and Bordetella parapertussis in mice. Here, we show that a single nasal administration of BPZE1 protects mice against lethal infection with B. bronchiseptica. After challenge, the vaccinated animals displayed markedly reduced lung inflammation and tissue damage, decreased neutrophil infiltration and increased levels of CD4(+)CD25(+)FoxP3(+) regulatory T cells in the lungs compared to non-immunized mice. Depletion of these cells abolished BPZE1-induced protection, indicating that BPZE1 protects against lethal inflammation through the recruitment of regulatory T cells. In addition, the B. bronchiseptica load was significantly decreased in the vaccinated animals. Using passive transfer experiments, protection was found to be essentially cell mediated, and BPZE1-induced Th1 and Th17 T cells recognize whole B. bronchiseptica extracts, although the participation of antibodies in protection cannot be discounted. Thus, a single administration of BPZE1 can confer protection against B. bronchiseptica in mice by a dual mechanism.

Differential contribution of the repeats to heparin binding of HBHA, a major adhesin of Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis remains one of the most important causes of global mortality and morbidity, and the molecular mechanisms of the pathogenesis are still incompletely understood. Only few virulence factors of the causative agent Mycobacterium tuberculosis are known. One of them is the heparin-binding haemagglutinin (HBHA), an important adhesin for epithelial cells and an extrapulmonary dissemination factor. HBHA mediates mycobacterial adherence to epithelial cells via the interactions of its C-terminal, lysine rich repeat domain with sulfated glycoconjugates on the surface of epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Using defined heparin sulfate (HS) analogs, we determined the minimal heparin fragment length for HBHA binding and structural adaptations of the HBHA heparin-binding domain (HBD) upon binding to heparin. The NMR studies show significant shifts of all residues in the HBD upon interaction with heparin, with stronger shifts in the last repeats compared to the upstream repeats, and indicated that the HS fragments with 14 sugar units cover the entire C-terminal lysine-rich domain of HBHA. The differential implication of the repeats is determined by the relative position of prolines and lysines within each repeat, and may contribute to binding specificity. GAG binding induces a non-homogeneous structural rearrangement in the HBD, with stabilization of a nascent α-helix only in the last penta-repeats. CONCLUSION/SIGNIFICANCE: Mycobacterial HBHA undergoes structural adaptation upon interaction with GAGs, which is likely involved in binding specificities of the adhesin, and mycobacterial pathogens may use HBD polymorphisms for host or organ specificity. Further studies will aim at decoding the complementarity between HBD repeats and HS sequence.

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence.

When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease.

Mycobacterium avium subsp. paratuberculosis (Map) causes a chronic enteric disease in ruminants, called paratuberculosis or Johne's disease. The current model proposes that after ingestion by the host, Map crosses the intestinal barrier via internalization by the M cells. Experimental observations suggest, however, that Map may also transcytose the intestinal wall via the enterocytes, but the mechanisms involved in this process remain poorly understood. Cytoadherence assays performed on epithelial cells with Map revealed that the addition of laminin to the cell culture increases adhesion. A Map protein was isolated by heparin-Sepharose chromatography and identified as a laminin-binding protein like. The gene encoding this protein named Lbp/Hlp was identified in the Map genome sequence at locus MAP3024 (annotated Hup B). The deduced Map Lbp/Hlp amino acid sequence reveals 80% identity with that reported for other mycobacteria. The C-terminal domain involved in adhesion is mainly composed of arginine and lysine residues modified by methylation. In vitro tests demonstrated that recombinant Lbp/Hlp binds laminin, heparin, collagen and epithelial cells. Interestingly, we found that this adhesin corresponds to the antigen described as the target of pANCA and serum antibodies of patients with Crohn's disease.