Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation.
ABSTRACT: Bacterial biofilms are complex microbial communities that are common in nature and are being recognized increasingly as an important determinant of bacterial virulence. However, the structural determinants of bacterial aggregation and eventual biofilm formation have been poorly defined. In Gram-negative bacteria, a major subgroup of extracellular proteins called self-associating autotransporters (SAATs) can mediate cell-cell adhesion and facilitate biofilm formation. In this study, we used the Haemophilus influenzae Hap autotransporter as a prototype SAAT to understand how bacteria associate with each other. The crystal structure of the H. influenzae Hap(S) passenger domain (harbouring the SAAT domain) was determined to 2.2 Å by X-ray crystallography, revealing an unprecedented intercellular oligomerization mechanism for cell-cell interaction. The C-terminal SAAT domain folds into a triangular-prism-like structure that can mediate Hap-Hap dimerization and higher degrees of multimerization through its F1-F2 edge and F2 face. The intercellular multimerization can give rise to massive buried surfaces that are required for overcoming the repulsive force between cells, leading to bacterial cell-cell interaction and formation of complex microcolonies.
Project description:Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or ?5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ?ompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.
Project description:Nontypeable Haemophilus influenzae is a major cause of localized respiratory tract disease and initiates infection by colonizing the nasopharynx. Colonization requires adherence to host epithelial cells, which is mediated by surface proteins such as the Hap adhesin. In this study, we identified a relationship between Hap levels in the outer membrane and lipopolysaccharide (LPS) biosynthesis enzymes. We found that mutation of the rfaF, pgmB, lgtC, kfiC, orfE, rfbP, lsgB, or lsgD genes, which are involved in the synthesis of the LPS oligosaccharide core in H. influenzae strain Rd/HapS243A, resulted in loss of Hap in the bacterial outer membrane and a decrease in hap transcript levels. In contrast, the same mutations had no effect on outer membrane localization of H. influenzae P5 or IgA1 protease or levels of p5 or iga1 transcripts, suggesting a Hap-specific effect. Elimination of the HtrA periplasmic protease resulted in a return of Hap to the outer membrane and restoration of hap transcript levels. Consistently, in lgtC phase-off bacteria, Hap was absent from the outer membrane, and hap transcript levels were reduced. Hap localization and hap transcript levels were not related to LPS size but to the functions of the LPS biosynthesis enzymes themselves. We speculate that the lack of certain LPS biosynthesis enzymes causes Hap to mislocalize and accumulate in the periplasm, where it is degraded by HtrA. This degradation then leads to a decrease in hap transcript levels. Together, these data highlight a novel interplay between Hap and LPS biosynthesis that can influence H. influenzae interactions with the host.
Project description:BACKGROUND:Nontypeable Haemophilus influenzae (NTHi) is a common cause of respiratory tract disease and initiates infection by colonization in nasopharynx. The Haemophilus influenzae (H. influenzae) Hap adhesin is an auto transporter protein that promotes initial interaction with human epithelial cells. Hap protein contains a 110 kDa internal passenger domain called "HapS" and a 45 kDa C-terminal translocator domain called "Hap?". Hap adhesive activity has been recently reported to be connected to its Cell Binding Domain (CBD) which resides within the 311 C-terminal residues of the internal passenger domain of the protein. Furthermore, immunization with this CBD protein has been shown to prevent bacterial nasopharynx colonization in animal models. METHODS:To provide enough amounts of pure HapS protein for vaccine studies, we sought to develop a highly optimized system to overexpress and purify the protein in large quantities. To this end, pET24a-cbd plasmid harboring cbd sequence from NTHi ATCC49766 was constructed and its expression was optimized by testing various expression parameters such as growth media, induction temperature, IPTG inducer concentration, induction stage and duration. SDS-PAGE and Western-blotting were used for protein analysis and confirmation and eventually the expressed protein was easily purified via immobilized metal affinity chromatography (IMAC) using Ni-NTA columns. RESULTS:The highest expression level of target protein was achieved when CBD expressing E. coli BL21 (DE3) cells were grown at 37°C in 2xTY medium with 1.0 mM IPTG at mid-log phase (OD600 nm equal to 0.6) for 5 hrs. Amino acid sequence alignment of expressed CBD protein with 3 previously published CBD amino acid sequences were more than %97 identical and antigenicity plot analysis further revealed 9 antigenic domains which appeared to be well conserved among different analyzed CBD sequences. CONCLUSION:Due to the presence of high similarity among CBD from NTHi ATCC49766 and other NTHi strains, CBD protein expressed here sounds to be theoretically ideal as a universal candidate for being used in vaccine studies against NTHi strains of various geographical areas. Further investigations to corroborate the potency of this protein as a vaccine candidate are under process.
Project description:Haemophilus influenzae is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hap autotransporter protein mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells and presumably facilitates colonization. The serine protease activity of Hap is associated with autoproteolytic cleavage and extracellular release of the HapS passenger domain, leaving the Hapbeta C-terminal domain embedded in the outer membrane. Cleavage occurs most efficiently at the LN1036-37 peptide bond and to a lesser extent at three other sites. In this study, we utilized site-directed mutagenesis, homology modeling, and assays with a peptide library to characterize the structural determinants of Hap proteolytic activity and cleavage specificity. In addition, we used homology modeling to predict the S1, S2, and S4 subsite residues of the Hap substrate groove. Our results indicate that the P1 and P2 positions at the Hap cleavage sites are critical for cleavage, with leucine preferred over larger hydrophobic residues or other amino acids in these positions. The substrate groove is formed by L263 and N274 at the S1 subsite, R264 at the S2 subsite, and E265 at the S4 subsite. This information may facilitate design of approaches to block Hap activity and interfere with H. influenzae colonization.
Project description:Hospital-acquired bacterial infections are an increasingly important cause of morbidity and mortality worldwide. Staphylococcal species are responsible for the majority of hospital-acquired infections, which are often complicated by the ability of staphylococci to grow as biofilms. Biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus requires cell-surface proteins (Aap and SasG) containing sequence repeats known as G5 domains; however, the precise role of these proteins in biofilm formation is unclear. We show here, using analytical ultracentrifugation (AUC) and circular dichroism (CD), that G5 domains from Aap are zinc (Zn(2+))-dependent adhesion modules analogous to mammalian cadherin domains. The G5 domain dimerizes in the presence of Zn(2+), incorporating 2-3 Zn(2+) ions in the dimer interface. Tandem G5 domains associate in a modular fashion, suggesting a "zinc zipper" mechanism for G5 domain-based intercellular adhesion in staphylococcal biofilms. We demonstrate, using a biofilm plate assay, that Zn(2+) chelation specifically prevents biofilm formation by S. epidermidis and methicillin-resistant S. aureus (MRSA). Furthermore, individual soluble G5 domains inhibit biofilm formation in a dose-dependent manner. Thus, the complex three-dimensional architecture of staphylococcal biofilms results from the self-association of a single type of protein domain. Surface proteins with tandem G5 domains are also found in other bacterial species, suggesting that this mechanism for intercellular adhesion in biofilms may be conserved among staphylococci and other Gram-positive bacteria. Zn(2+) chelation represents a potential therapeutic approach for combating biofilm growth in a wide range of bacterial biofilm-related infections.
Project description:Novel strategies in the field of nanotechnology for the development of suitable multifunctional drug delivery vehicles have been pursued with promising upshots. Luminescent copper nanocluster-doped hydroxyapatite nanoparticles (HAP NPs) were synthesized and applied for the delivery of antibacterial drug kanamycin. The negatively charged doped HAP NPs could electrostatically interact with the positively charged kanamycin. The kanamycin-loaded doped HAP NPs showed pronounced activity in the case of Gram-negative bacteria compared to that in Gram-positive bacteria. Upon interaction with the bacteria, kanamycin could probably generate harmful agents such as hydroxyl radical that leads to bacterial cell damage. After being incorporated with copper nanoclusters (Cu NCs), the doped HAP NPs were applied for the bioimaging of bacterial cells. The biocompatibility of doped HAP NPs was also studied in HeLa cells. As compared to copper nanoclusters, the doped HAP NPs showed excellent biocompatibility even at higher concentrations of copper. The kanamycin-loaded doped HAP NPs were further applied toward Pseudomonas aeruginosa biofilm eradication. Thus, the as-synthesized copper nanocluster-doped HAP NPs were applied as nanocarriers for antibiotic drug delivery, bioimaging, and antibiofilm applications.
Project description:LytM proteins belong to a family of bacterial metalloproteases. In Gram-negative bacteria, LytM factors are mainly reported to have a direct effect on cell division by influencing cleavage and remodeling of peptidoglycan. In this study, mining nontypeable Haemophilus influenzae (NTHI) genomes, three highly conserved open reading frames (ORFs) containing a LytM domain were identified, and the proteins encoded by the ORFs were named YebA, EnvC, and NlpD on the basis of their homology with the Escherichia coli proteins. Immunoblotting and confocal analysis showed that while NTHI NlpD is exposed on the bacterial surface, YebA and EnvC reside in the periplasm. NTHI ?yebA and ?nlpD deletion mutants revealed an aberrant division phenotype characterized by an altered cell architecture and extensive membrane blebbing. The morphology of the ?envC deletion mutant was identical to that of the wild-type strain, but it showed a drastic reduction of periplasmic proteins, including the chaperones HtrA, SurA, and Skp, and an accumulation of ?-barrel-containing outer membrane proteins comprising the autotransporters Hap, IgA serine protease, and HMW2A, as observed by proteomic analysis. These data suggest that EnvC may influence the bacterial surface protein repertoire by facilitating the passage of the periplasmic chaperones through the peptidoglycan layer to the close vicinity of the inner face of the outer membrane. This hypothesis was further corroborated by the fact that an NTHI envC defective strain had an impaired capacity to adhere to epithelial cells and to form biofilm. Notably, this strain also showed a reduced serum resistance. These results suggest that LytM factors are not only important components of cell division but they may also influence NTHI physiology and pathogenesis by affecting membrane composition.Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen that colonizes the human nasopharynx and can cause serious infections in children (acute otitis media) and adults (chronic obstructive pulmonary disease). Several virulence factors are well studied, but the complete scenario of NTHI pathogenesis is still unclear. We identified and characterized three NTHI LytM factors homologous to the Escherichia coli LytM proteins. Although LytM factors are reported to play a crucial role in the cell division process, in NTHI they are also involved in other bacterial functions. In particular, YebA and NlpD are fundamental for membrane stability: indeed, their absence causes an increased release of outer membrane vesicles (OMVs). On the other hand, our data suggest that EnvC could directly or indirectly affect peptidoglycan permeability and consequently, bacterial periplasmic and outer membrane protein distribution. Interestingly, by modulating the surface composition of virulence determinants, EnvC also has an impact on NTHI pathogenesis.
Project description:The periodontal pathogen Actinobacillus actinomycetemcomitans possesses myriad virulence factors, among them the ability to adhere to and invade epithelial cells. Recent advances in the molecular manipulation of this pathogen and the sequencing of strain HK 1651 (http://www.genome.ou.edu/act.html) have facilitated examination of the genetics of its interaction with epithelial cells. The related gram-negative organism, Haemophilus influenzae, possesses autotransporter adhesins. A search of the sequence database of strain HK 1651 revealed a homologue with similarity in the pore-forming domain to that of the H. influenzae autotransporter, Hap. A. actinomycetemcomitans mutants deficient in the homologue, Aae, showed reduced binding to epithelial cells. A method for making A. actinomycetemcomitans SUNY 465 transiently resistant to spectinomycin was used with conjugation to generate an isogenic aae mutant. An allelic replacement mutant was created in the naturally transformable A. actinomycetemcomitans strain ATCC 29523. Lactoferrin, an important part of the innate host defense system, protects against bacterial infection by bactericidal and antiadhesion mechanisms. Lactoferrin in human milk removes or cleaves Hap and another autotransporter, an immunoglobulin A1 protease, from the surface of H. influenzae, thereby reducing their binding to epithelial cells. Human milk whey had similar effects on Aae from A. actinomycetemcomitans ATCC 29523 and its binding to epithelial cells; however, there was little effect on the binding of SUNY 465. A difference in the genetic structure of aae in the two strains, apparently due to the copy number of a 135-base repeated sequence, may be the cause of the differential action of lactoferrin. aae is the first A. actinomycetemcomitans gene involved in adhesion to epithelial cells to be identified.
Project description:Otitis media with effusion (OME) is a biofilm driven disease and commonly accepted otopathogens, such as Haemophilus influenzae, Streptococcus pneumonia, and Moraxella catarrhalis, have been demonstrated to form polymicrobial biofilms within the middle ear cleft. However, Alloiococcus otitidis (A. otitidis), which is one of the most commonly found bacteria within middle ear aspirates of children with OME, has not been described to form biofilms. The aim of this study was to investigate whether A. otitidis can form biofilms and investigate the impact on antibiotic susceptibility and survivability in polymicrobial biofilms with H. influenzae in vitro. The ability of A. otitidis to form single-species and polymicrobial biofilms with H. influenzae was explored. Clinical and commercial strains of A. otitidis and H. influenzae were incubated in brain heart infusion with and without supplementation. Biofilm was imaged using confocal laser scanning microscopy and scanning electron microscopy. Quantification of biofilm biomass and viable bacterial number was assessed using crystal violet assays and viable cell counting in both optimal growth conditions and in adverse growth conditions (depleted media and sub-optimal growth temperature). Antimicrobial susceptibility and changes in antibiotic resistance of single-species and multi-species co-culture were assessed using a microdilution method to assess minimal bactericidal concentration and E-test for amoxicillin and ciprofloxacin. A. otitidis formed single-species and polymicrobial biofilms with H. influenzae. Additionally, whilst strain dependent, combinations of polymicrobial biofilms decreased antimicrobial susceptibility, albeit a small magnitude, in both planktonic and polymicrobial biofilms. Moreover, A. otitidis promoted H. influenzae survival by increasing biofilm production in depleted media and at suboptimal growth temperature. Our findings suggest that A. otitidis may play an indirect pathogenic role in otitis media by altering H. influenzae antibiotic susceptibility and enhancing growth under adverse conditions.
Project description:Biofilms contribute to the pathogenesis of many forms of Staphylococcus aureus infection. Treatment of these infections is complicated by intrinsic resistance to conventional antibiotics, thus creating an urgent need for strategies that can be used for the prevention and treatment of biofilm-associated infections.This study demonstrates that a botanical natural product composition (220D-F2) rich in ellagic acid and its derivatives can limit S. aureus biofilm formation to a degree that can be correlated with increased antibiotic susceptibility. The source of this composition is Rubus ulmifolius Schott. (Rosaceae), a plant used in complementary and alternative medicine in southern Italy for the treatment of skin and soft tissue infections. All S. aureus clonal lineages tested exhibited a reduced capacity to form a biofilm at 220D-F2 concentrations ranging from 50-200 µg/mL, which were well below the concentrations required to limit bacterial growth (530-1040 µg/mL). This limitation was therapeutically relevant in that inclusion of 220D-F2 resulted in enhanced susceptibility to the functionally-distinct antibiotics daptomycin, clindamycin and oxacillin. Testing with kidney and liver cell lines also demonstrated a lack of host cell cytotoxicity at concentrations of 220D-F2 required to achieve these effects.These results demonstrate that extract 220D-F2 from the root of Rubus ulmifolius can be used to inhibit S. aureus biofilm formation to a degree that can be correlated with increased antibiotic susceptibility without toxic effects on normal mammalian cells. Hence, 220D-F2 is a strong candidate for development as a botanical drug for use in the prevention and treatment of S. aureus biofilm-associated infections.