trans-Endothelial neutrophil migration activates bactericidal function via Piezo1 mechanosensing.

The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.

Dihydrolipoamide dehydrogenase of Pseudomonas aeruginosa is a surface-exposed immune evasion protein that binds three members of the factor H family and plasminogen.

The opportunistic human pathogen Pseudomonas aeruginosa causes a wide range of diseases. To cross host innate immune barriers, P. aeruginosa has developed efficient strategies to escape host complement attack. In this study, we identify the 57-kDa dihydrolipoamide dehydrogenase (Lpd) as a surface-exposed protein of P. aeruginosa that binds the four human plasma proteins, Factor H, Factor H-like protein-1 (FHL-1), complement Factor H-related protein 1 (CFHR1), and plasminogen. Factor H contacts Lpd via short consensus repeats 7 and 18-20. Factor H, FHL-1, and plasminogen when bound to Lpd were functionally active. Factor H and FHL-1 displayed complement-regulatory activity, and bound plasminogen, when converted to the active protease plasmin, cleaved the chromogenic substrate S-2251 and the natural substrate fibrinogen. The lpd of P. aeruginosa is a rather conserved gene; a total of 22 synonymous and 3 nonsynonymous mutations was identified in the lpd gene of the 5 laboratory strains and 13 clinical isolates. Lpd is surface exposed and contributes to survival of P. aeruginosa in human serum. Bacterial survival was reduced when Lpd was blocked on the surface prior to challenge with human serum. Similarly, bacterial survival was reduced up to 84% when the bacteria was challenged with complement active serum depleted of Factor H, FHL-1, and CFHR1, demonstrating a protective role of the attached human regulators from complement attack. In summary, Lpd is a novel surface-exposed virulence factor of P. aeruginosa that binds Factor H, FHL-1, CFHR1, and plasminogen, and the Lpd-attached regulators are relevant for innate immune escape and most likely contribute to tissue invasion.

Functional recruitment of the human complement inhibitor C4BP to Yersinia pseudotuberculosis outer membrane protein Ail.

Ail is a 17-kDa chromosomally encoded outer membrane protein that mediates serum resistance (complement resistance) in the pathogenic Yersiniae (Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis). In this article, we demonstrate that Y. pseudotuberculosis Ail from strains PB1, 2812/79, and YPIII/pIB1 (serotypes O:1a, O:1b, and O:3, respectively) can bind the inhibitor of the classical and lectin pathways of complement, C4b-binding protein (C4BP). Binding was observed irrespective of serotype tested and independently of YadA, which is the primary C4BP receptor of Y. enterocolitica. Disruption of the ail gene in Y. pseudotuberculosis resulted in loss of C4BP binding. Cofactor assays revealed that bound C4BP is functional, because bound C4BP in the presence of factor I cleaved C4b. In the absence of YadA, Ail conferred serum resistance to strains PB1 and YPIII, whereas serum resistance was observed in strain 2812/79 in the absence of both YadA and Ail, suggesting additional serum resistance factors. Ail from strain YPIII/pIB1 alone can mediate serum resistance and C4BP binding, because its expression in a serum-sensitive laboratory strain of Escherichia coli conferred both of these phenotypes. Using a panel of C4BP mutants, each deficient in a single complement control protein domain, we observed that complement control protein domains 6-8 are important for binding to Ail. Binding of C4BP was unaffected by increasing heparin or salt concentrations, suggesting primarily nonionic interactions. These results indicate that Y. pseudotuberculosis Ail recruits C4BP in a functional manner, facilitating resistance to attack from complement.

Full functional activity of SSL7 requires binding of both complement C5 and IgA.

Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a range of diseases, from local skin infections through to life-threatening illnesses such as toxic shock syndrome. S. aureus produces an assortment of molecules designed to evade or subvert the host immune system. One example is the 23 kDa staphylococcal superantigen-like protein 7 (SSL7) that simultaneously binds immunoglobulin A (IgA) and complement C5 to inhibit complement-mediated hemolytic and bactericidal activity. The avirulent bacterium Lactococcus lactis was engineered to express SSL7 so that its role in bacterial survival could be assessed without interference from other virulence factors. Expression of SSL7 by L. lactis led to significantly enhanced bacterial survival in whole human blood and prevented the membrane attack complex (C5b-9) forming on the cell wall. To further understand the mechanism of action of SSL7, the activity of wild-type SSL7 protein was compared with a panel of mutant proteins lacking the capacity to bind IgA, C5, or both IgA and C5. SSL7 potently inhibited in vitro chemotaxis of inflammatory myeloid cells in response to a pathogenic stimulus and when injected into mice, SSL7 blocked the migration of neutrophils into the peritoneum in response to an inoculum of heat-killed S. aureus. Mutagenesis of the C5-binding site on SSL7 abolished all inhibitory activity, while mutation of the IgA-binding site had only partial effects, indicating that while IgA binding enhances activity it is not essential. SSL7 is an important staphylococcal virulence factor with potent anti-inflammatory properties, which are mediated by targeting complement C5 and IgA.

Molecular basis of increased serum resistance among pulmonary isolates of non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi), a common commensal of the human pharynx, is also an opportunistic pathogen if it becomes established in the lower respiratory tract (LRT). In comparison to colonizing isolates from the upper airway, LRT isolates, especially those associated with exacerbations of chronic obstructive pulmonary disease, have increased resistance to the complement- and antibody-dependent, bactericidal effect of serum. To define the molecular basis of this resistance, mutants constructed in a serum resistant strain using the mariner transposon were screened for loss of survival in normal human serum. The loci required for serum resistance contribute to the structure of the exposed surface of the bacterial outer membrane. These included loci involved in biosynthesis of the oligosaccharide component of lipooligosaccharide (LOS), and vacJ, which functions with an ABC transporter encoded by yrb genes in retrograde trafficking of phospholipids from the outer to inner leaflet of the cell envelope. Mutations in vacJ and yrb genes reduced the stability of the outer membrane and were associated with increased cell surface hyrophobicity and phospholipid content. Loss of serum resistance in vacJ and yrb mutants correlated with increased binding of natural immunoglobulin M in serum as well as anti-oligosaccharide mAbs. Expression of vacJ and the yrb genes was positively correlated with serum resistance among clinical isolates. Our findings suggest that NTHi adapts to inflammation encountered during infection of the LRT by modulation of its outer leaflet through increased expression of vacJ and yrb genes to minimize recognition by bactericidal anti-oligosaccharide antibodies.

CC chemokine ligand 3 overcomes the bacteriocidal and phagocytic defect of macrophages and hastens recovery from experimental otitis media in TNF-/- mice.

Innate immune mechanisms are crucial in defense against bacterial illnesses in humans, as evidenced by abnormal antibacterial responses due to defects in TLR signaling, seen in children with MyD88 or IL-1R-associated kinase 4 deficiency. Otitis media (OM) is the most common disease of childhood, and the role of innate immune molecules in this disorder remains unclear. In a murine model of OM, we show that, in the absence of TNF, a key effector of innate immunity, this disease is prolonged after middle ear infection with nontypeable Haemophilus influenzae (NTHi). In the absence of TNF, mice fail to upregulate both TLRs and downstream genes and proteins, such as CCL3, resulting in defects in both inflammatory cell recruitment and macrophage function. Peritoneal macrophages of mice lacking TNF have a diminished ability to phagocytose and kill NTHi, and this defect is partially corrected in vitro by exogenous rTNF. Addition of rCCL3 alone or in combination with rTNF restores phagocytosis and killing by TNF-deficient macrophages to that of unstimulated wild-type macrophages. In vivo administration of rCCL3 to animals deficient in TNF fully restores the ability to control OM due to NTHi, whereas a CCL3-blocking Ab impaired the ability of wild-type mice to recover from OM. Thus, CCL3 is a potent downstream effector of TNF-mediated inflammation in vitro and in vivo. Manipulation of CCL3 and/or TNF may prove to be effective therapeutic approaches in OM or other conditions associated with defective TNF generation.

Differential analysis of bactericidal systems of blood serum with recombinant luminescent Escherichia coli and Bacillus subtilis strains.

Luminescence intensity of recombinant Escherichia coli and Bacillus subtilis strains with cloned luxCD(AB)E genes of the natural luminescent microorganism Photobacterium leiognathi was studied under the influence of 30 individual samples of human blood serum of different component composition. A relationship was found between the level of residual bioluminescence and degree of the bactericidal effect. Moreover, the inhibition of E. coli lux+ luminescence was shown to be related to activity of the complement-lysozyme system. The reaction of B. subtilis lux+ primarily depended on the presence of ß-lysin in the blood serum. These data provide an experimental substantiation of a new method of differential analysis of humoral factors of nonspecific innate immunity with recombinant luminescent bacteria.

Nontypeable Haemophilus influenzae protein E binds vitronectin and is important for serum resistance.

Nontypeable Haemophilus influenzae (NTHi) commonly causes local disease in the upper and lower respiratory tract and has recently been shown to interfere with both the classical and alternative pathways of complement activation. The terminal pathway of the complement system is regulated by vitronectin that is a component of both plasma and the extracellular matrix. In this study, we identify protein E (PE; 16 kDa), which is a recently characterized ubiquitous outer membrane protein, as a vitronectin-binding protein of NTHi. A PE-deficient NTHi mutant had a markedly reduced survival in serum compared with the PE-expressing isogenic NTHi wild type. Moreover, the PE-deficient mutant showed a significantly decreased binding to both soluble and immobilized vitronectin. In parallel, PE-expressing Escherichia coli bound soluble vitronectin and adhered to immobilized vitronectin compared with controls. Surface plasmon resonance technology revealed a K(D) of 0.4 microM for the interaction between recombinant PE and immobilized vitronectin. Moreover, the PE-dependent vitronectin-binding site was located at the heparin-binding domains of vitronectin and the major vitronectin-binding domain was found in the central core of PE (aa 84-108). Importantly, vitronectin bound to the surface of NTHi 3655 reduced membrane attack complex-induced hemolysis. In contrast to incubation with normal human serum, NTHi 3655 showed a reduced survival in vitronectin-depleted human serum, thus demonstrating that vitronectin mediates a protective role at the bacterial surface. Our findings show that PE, by binding vitronectin, may play an important role in NTHi pathogenesis.

Binding of complement inhibitor C4b-binding protein contributes to serum resistance of Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the alpha-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.

Designed beta-sheet-forming peptide 33mers with potent human bactericidal/permeability increasing protein-like bactericidal and endotoxin neutralizing activities.

Novel peptide 33mers have been designed by incorporating beta-conformation stabilizing residues from the beta-sheet domains of alpha-chemokines and functionally important residues from the beta-sheet domain of human neutrophil bactericidal protein (B/PI). B/PI is known for its ability to kill bacteria and to neutralize the action of bacterial endotoxin (lipopolysaccharide, LPS) which can induce septic shock leading to eventual death. Here, the goal was to make short linear peptides which demonstrate good beta-sheet folding and maintain bioactivity as in native B/PI. A library of 24 peptide 33mers (betapep-1 to betapep-24) were synthesized with various amino acid substitutions. CD and NMR data acquired in aqueous solution indicate that betapep peptides form beta-sheet structure to varying degrees and self-associate as dimers and tetramers like the alpha-chemokines. Bactericidal activity toward Gram-negative Pseudomonas aeruginosa was tested, and betapep-19 was found to be only about 5-fold less potent (62% kill at 1.2 x 10(-7) M) than native B/PI (80% kill at 2.9 x 10(-8) M). At LPS neutralization, betapep-2 and -23 were found to be most active (66-78% effective at 1.2 x 10(-6) M), being only about 50-100-fold less active than B/PI (50% at 1.5 x 10(-8) M). In terms of structure-activity relations, beta-sheet structural stability correlates with the capacity to neutralize LPS, but not with bactericidal activity. Although a net positive charge is necessary for activity, it is not sufficient for optimal activity. Hydrophobic residues tend to influence activities indirectly by affecting structural stability. Furthermore, results show that sequentially and spatially related residues from the beta-sheet domain of native B/PI can be designed into short linear peptides which show good beta-sheet folding and retain much of the native activity. This research contributes to the development of solutions to the problem of multiple drug-resistant, opportunistic microorganisms like P. aeruginosa and of agents effective at neutralizing bacterial endotoxin.

Genetic resistance/susceptibility to mycobacteria: phenotypic expression in bone marrow derived macrophage lines.

Congenic strains of mice susceptible (B10A.Bcgs) or resistant (B10A.Bcgr) to BCG were established. Here we describe the model system which has been established to analyze the functional activities of macrophages in the two strains. We have immortalized bone marrow macrophages from B10A.Bcgs and B10A.Bcgr congenic strains of mice and derived cloned macrophage lines designated B10S and B10R, respectively. B10R and B10S cell lines exhibited surface markers and morphology typical of macrophages. B10S and B10R were similar in their phagocytic activity, in their level of c-fms, in their transforming growth factor beta (TGF beta) mRNAs expression, and in their expression of tumoricidal activity in response to interferon-gamma (IFN gamma) plus lipopolysaccharides (LPS). However, B10R macrophages expressed a higher level of la mRNA when activated with IFN gamma compared with B10S macrophages. Analysis of the bacteriostatic activity of the two cell lines revealed that B10R macrophages were much more active in inhibiting Mycobacterium smegmatis replication than B10S. To measure the intracellular destruction of bacilli, a bactericidal assay based on hybridization with an oligonucleotide probe specific for mycobacterial ribosomal RNA was designed. The results demonstrated that B10R macrophages were endowed with enhanced constitutive bactericidal activity as compared with B10S. In conclusion we have obtained macrophage lines from bone marrow of B10A.Bcgs and B10A.Bcgr mice that express to a similar extent functional and phenotypic characteristics of macrophages. However, we demonstrate that relative to B10S macrophages, the B10R macrophages have higher expression of la mRNA and that they are constitutively more active in expressing mycobactericidal activity.

Intracellular survival of Brucella abortus, Mycobacterium bovis BCG, Salmonella dublin, and Salmonella typhimurium in macrophages from cattle genetically resistant to Brucella abortus.

Peripheral blood monocyte-derived macrophages were obtained from a herd of cows selected, bred, and confirmed as resistant or susceptible by in vivo challenge of Brucella abortus Strain 2308. The ability to control in vitro intracellular bacterial replication of B. abortus Strain 2308, Mycobacterium bovis Bacillus Calmette-Guerin (BCG) Montreal Strain 9003, Salmonella dublin Strain 5631, and Salmonella typhimurium Strain 14028 was evaluated in a bactericidal assay. The macrophages from resistant cattle were significantly superior (P < 0.05) in controlling intracellular growth of B. abortus, M. bovis BCG, S. dublin but not of S, typhimurium than macrophages from susceptible animals. Controls of all four pathogens correlated strongly with each other in resistant or susceptible macrophages. Data from resistant cattle had a tighter grouping than that of susceptible cattle, while data from susceptible cattle overlapped considerably with data from resistant animals. Therefore, this assay was considered a phenotypic marker of the resistant trait. For each bacterial species a percent bacterial survival value was used as a cut-off point to designate animals as resistant or susceptible. These data were compared with the in vivo challenged resistant or susceptible classification by using the Chi-square analyses. A cut-off point of 70 percent bacterial survival for B. abortus designated 14 cattle as susceptible and seven as resistant and this correlated 100 percent with the number of animals designated as to the relevant category by in vivo challenge. A value of 65 percent bacterial survival for M. bovis BCG, and 100 percent bacterial survival for S. dublin correlated highly with actual numbers of animals designated as susceptible or resistant.

The influence of genetic variation on the splenic T cell cytokine and specific serum antibody responses to Porphyromonas gingivalis in mice.

BACKGROUND: T cell cytokine profiles in the spleens and anti-Porphyromonas gingivalis antibodies in the sera of P. gingivalis-immunized BALB/c (H-2d), CBA/CaH (H-2k), C57BL6 (H-2b), and DBA/2J (H-2d, C5 deficient) mice were examined. METHODS: Mice were immunized either by intraperitoneal injections of P. gingivalis outer membrane antigens and Freund's incomplete adjuvant weekly for 3 weeks or sham-immunized with PBS and adjuvant, followed by subcutaneous challenge with live organisms 1 week after the final immunization. Spleens were excised and blood samples collected by heart puncture at 0 and 7 days after challenge. Splenic CD4 and CD8 cells were stained for intracytoplasmic interleukin (IL)-4, interferon (IF)-gamma, and IL-10 and levels of anti-P. gingivalis antibodies in the serum samples determined by ELISA. RESULTS: Lesion sizes in immunized BALB/c mice remained stable for the 7-day experimental period. Immunized CBA/CaH and C57BL6 mice exhibited large lesions at day 1 reducing by day 7 particularly in the latter strain. Lesions in immunized DBA/2J mice were still larger than the other strains at day 7. With the exception of DBA/2J mice, sham-immunized mice demonstrated lesions which did not show signs of healing by day 7. T cell cytokine responses in sham-immunized mice at day 0 were low, increasing to a variable degree by day 7 after challenge in the 4 strains. Immunized BALB/c mice demonstrated intermediate T cell responses while generally exhibiting a stronger IFN-gamma response than IL-4 or IL-10. Immunized CBA/CaH and C57BL6 mice showed weak T cell cytokine responses while immunized DBA/2J displayed the strongest T cell responses particularly in regard to IL-4 positive cells. Sham-immunized mice had low levels of serum anti-P. gingivalis antibody levels at day 0 with levels increasing significantly by day 7 after challenge. Antibody levels in immunized mice seemed to correlate with lesion sizes. Immunized C57BL6 mice had the highest antibody levels followed by CBA/CaH, BALB/c with DBA/2J exhibiting low levels. The T cell and B cell antibody responses in each strain appeared to exhibit an inverse relationship. CONCLUSIONS: This study has shown that genetic differences at the level of H-2 haplotype induce variations in the local and T and B cell responses to P. gingivalis antigens. The responses of DBA/2J mice which have the same haplotype as BALB/c mice suggest that factors other than H-2 haplotype such as the C5 deficiency may influence this immune response. The significance of the specific antibody and T cell responses and of their inverse relationship to susceptibility to periodontal disease remains to be determined.

Identification of the primary mechanism of complement evasion by the periodontal pathogen, Treponema denticola.

Treponema denticola, a periodontal pathogen, binds the complement regulatory protein Factor H (FH). Factor H binding protein B (FhbB) is the sole FH binding protein produced by T. denticola. The interaction of FhbB with FH is unique in that FH is bound to the cell and then cleaved by the T. denticola protease, dentilisin. A ∼ 50-kDa product generated by dentilisin cleavage is retained at the cell surface. Until this study, a direct role for the FhbB-FH interaction in complement evasion and serum sensitivity had not been demonstrated. Here we assess the serum resistance of T. denticola strain 35405 (Td35405wt) and isogenic mutants deficient in dentilisin (Td35405-CCE) and FhbB production (Td35405ΔfhbB), respectively. Both dentilisin and FhbB have been postulated to be key virulence factors that mediate complement evasion. Consistent with conditions in the subgingival crevice, an environment with a significant concentration of complement, Td35405wt was resistant to serum concentrations as high as 25%. Deletion of fhbB (Td35405ΔfhbB), which resulted in the complete loss of FH binding ability, but not inactivation of dentilisin activity (Td35405-CCE), rendered T. denticola highly sensitive to 25% human serum with 80% of the cells being disrupted after 4 h of incubation. Heat treatment of the serum to inactivate complement confirmed that killing was mediated by complement. These results indicate that the FH-FhbB interaction is required for serum resistance whereas dentilisin is not. This report provides new insight into the novel complement evasion mechanisms of T. denticola.

[Study on the classification of gonococcal island of different genotypes and effects of sac-4 gene on serum resistance of Neisseria gonorrhoeae].

OBJECTIVE: Using molecular methods to study the relationship between genotypes and serum resistance of Neisseria gonorrhoeae in Wuhan area. METHODS: NG-mast and serum bactericidal assays at the molecular level were used to differentiate the 46 strains which were isolated from the outpatients of sexually transmitted disease clinics and the relationship between different genotypes while phenotypes was also studied. RESULTS: 80.43% of the 46 strains contained the island and we were able to define three different combinations of genes in the isolates. Results from serum bactericidal assays showed that all 9 sac-4+ strains did not provide any serum resistance. CONCLUSION: Different isolates carried different gonococcal genetic islands (pathogenicity island) and certain phenotypes. There were no sobious relationship between sac-4 gene and serum resistance of Neisseria gonorrhoeae.

Secondary acylation of Klebsiella pneumoniae lipopolysaccharide contributes to sensitivity to antibacterial peptides.

Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055DeltalpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055DeltalpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins.

Haemophilus influenzae surface fibrils contribute to serum resistance by interacting with vitronectin.

Vitronectin inhibits the membrane attack complex of the complement system and is found both in plasma and the extracellular matrix. In this study, we have identified the outer membrane protein Haemophilus surface fibrils (Hsf) as the major vitronectin-binding protein in encapsulated H. influenzae type b. A H. influenzae mutant devoid of Hsf showed a significantly decreased binding to both soluble and immobilized vitronectin as compared with the wild-type counterpart. Moreover, Escherichia coli-expressing Hsf at the surface strongly adhered to immobilized vitronectin. Importantly, the H. influenzae Hsf mutant had a markedly reduced survival as compared with the wild-type bacterium when incubated with normal human serum. A series of truncated Hsf fragments were recombinantly manufactured in E. coli. The vitronectin binding regions were located within two separate binding domains. In conclusion, Hsf interacts with vitronectin and thereby inhibits the complement-mediated bactericidal activity, and thus is a major H. influenzae virulence factor.

The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance.

Neisseria meningitidis binds factor H (fH), a key regulator of the alternative complement pathway. A approximately 29 kD fH-binding protein expressed in the meningococcal outer membrane was identified by mass spectrometry as GNA1870, a lipoprotein currently under evaluation as a broad-spectrum meningococcal vaccine candidate. GNA1870 was confirmed as the fH ligand on intact bacteria by 1) abrogation of fH binding upon deleting GNA1870, and 2) blocking fH binding by anti-GNA1870 mAbs. fH bound to whole bacteria and purified rGNA1870 representing each of the three variant GNA1870 families. We showed that the amount of fH binding correlated with the level of bacterial GNA1870 expression. High levels of variant 1 GNA1870 expression (either by allelic replacement of gna1870 or by plasmid-driven high-level expression) in strains that otherwise were low-level GNA1870 expressers (and bound low amounts of fH by flow cytometry) restored high levels of fH binding. Diminished fH binding to the GNA1870 deletion mutants was accompanied by enhanced C3 binding and increased killing of the mutants. Conversely, high levels of GNA1870 expression and fH binding enhanced serum resistance. Our findings support the hypothesis that inhibiting the binding of a complement down-regulator protein to the neisserial surface by specific Ab may enhance intrinsic bactericidal activity of the Ab, resulting in two distinct mechanisms of Ab-mediated vaccine efficacy. These data provide further support for inclusion of this molecule in a meningococcal vaccine. To reflect the critical function of this molecule, we suggest calling it fH-binding protein.

The peptidoglycan-degrading property of lysozyme is not required for bactericidal activity in vivo.

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in pulmonary host defense. Increased concentration of lysozyme in the airspaces of transgenic mice enhanced bacterial killing whereas lysozyme deficiency resulted in increased bacterial burden and morbidity. Lysozyme degrades peptidoglycan in the bacterial cell wall leading to rapid killing of Gram-positive organisms; however, this mechanism cannot account for the protective effect of lysozyme against Gram-negative bacteria. The current study was therefore designed to test the hypothesis that the catalytic activity (muramidase activity) of lysozyme is not required for bacterial killing in vivo. Substitution of serine for aspartic acid at position 53 (D53S) in mouse lysozyme M completely ablated muramidase activity. Muramidase-deficient recombinant lysozyme (LysM(D53S)) killed both Gram-positive and Gram-negative bacteria in vitro. Targeted expression of LysM(D53S) in the respiratory epithelium of wild-type (LysM(+/+)/LysM(D53S)) or lysozyme M(null) mice (LysM(-/-)/LysM(D53S)) resulted in significantly elevated lysozyme protein in the airspaces without any increase in muramidase activity. Intratracheal challenge of transgenic mice with Gram-positive or Gram-negative bacteria resulted in a significant increase in bacterial burden in LysM(-/-) mice that was completely reversed by targeted expression of LysM(D53S). These results indicate that the muramidase activity of lysozyme is not required for bacterial killing in vitro or in vivo.

Identification of the outer-membrane protein PagC required for the serum resistance phenotype in Salmonella enterica serovar Choleraesuis.

Serum resistance is a crucial virulence factor for the development of systemic infections, including bacteraemia, by many pathogenic bacteria. Salmonella enterica serovar Choleraesuis is an important enteric pathogen that causes serious systemic infections in swine and humans. Here, it was found that, when introduced into Escherichia coli, a recombinant plasmid carrying the pagC gene from a plasmid-based genomic library of S. enterica serovar Choleraesuis conferred a high-level resistance to the bactericidal activity of pooled normal swine serum. The resistance was equal to the level conferred by rck, a gene encoding a 17 kDa outer-membrane protein which promotes the serum resistance phenotype in S. enterica serovar Typhimurium. Insertional mutagenesis of the cloned pagC gene generated a mutation that resulted in the loss of the serum resistance phenotype in E. coli. When this mutation was introduced into the chromosome of S. enterica serovar Choleraesuis by homology recombination with the wild-type allele, the resulting strain could not produce PagC, and it showed a decreased level of resistance to complement-mediated killing. The mutation could be restored by introduction of the intact pagC gene on a plasmid, but not by introduction of the point-mutated pagC gene. In addition, PagC was able to promote serum resistance in the S. enterica serovar Choleraesuis LPS mutant strain, which is highly sensitive to serum killing. Although PagC is not thought to confer serum resistance directly, these results strongly suggest that PagC is an important outer-membrane protein that plays an important role in the serum resistance of S. enterica serovar Choleraesuis.