Absence of high molecular weight proteins 1 and/or 2 is associated with decreased adherence among non-typeable Haemophilus influenzae clinical isolates

2013 ◽  
Vol 62 (11) ◽  
pp. 1649-1656 ◽  
Author(s):  
Jeni Vuong ◽  
Xin Wang ◽  
Jordan M. Theodore ◽  
Jennifer Whitmon ◽  
Patricia Gomez de Leon ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Haemophilus Influenzae ◽  
High Molecular Weight ◽  
Surface Proteins ◽  
Clinical Isolates ◽  
Type Iv ◽  
High Adherence ◽  
Genes Encoding ◽  
Type Iv Pilin ◽  
Pilin Protein

High molecular weight (Hmw) proteins 1 and 2, type IV pilin protein (PilA), outer-membrane protein P5 (OmpP5), Haemophilus protein D (Hpd) and Haemophilus adhesive protein (Hap) are surface proteins involved in the adherence of non-typeable Haemophilus influenzae. One hundred clinical isolates were evaluated for the presence of the genes encoding these proteins by PCR and for their adherence capacity (AC) to Detroit 562 nasopharyngeal cells (D562). The majority of isolates were from blood (77/100); other sites were also represented. Confluent D562 monolayers (1.2×105 cells per well) were inoculated with standardized minimal infective doses (m.o.i.) of 102, 103 or 104 c.f.u. per well. The AC was categorized as low (<10 %) or high (≥10 %) depending on the percentage of c.f.u. adhering per well. All the isolates evaluated showed adherence: 69/100 (69 %) demonstrated high adherence, while 31/100 (31 %) showed low adherence. Of all the genes evaluated, hmw1A and/or hmw2A were detected in 69/100 (69 %) of isolates. The presence of hmw1A and/or hmw2A was associated with increased adherence to D562 cells (P≤0.001). Dot immunoblots were performed to detect protein expression using mAbs 3D6, AD6 and 10C5. Among the high-adherence isolates (n = 69), 72 % reacted with 3D6 and 21 % with 10C5. Our data indicate that the absence of Hmw1 and/or Hmw2 was associated with decreased adherence to D562 cells.

scholarly journals Biofilms Formed by Nontypeable Haemophilus influenzae In Vivo Contain both Double-Stranded DNA and Type IV Pilin Protein

2007 ◽  
Vol 189 (10) ◽  
pp. 3868-3875 ◽  
Author(s):  
Joseph A. Jurcisek ◽  
Lauren O. Bakaletz
Keyword(s):  
Haemophilus Influenzae ◽  
Outer Membrane Proteins ◽  
Mammalian Host ◽  
Type Iv ◽  
Double Stranded Dna ◽  
Type Iv Pilin ◽  
Pilin Protein ◽  
Biofilm Matrix

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) strains are members of the normal human nasopharyngeal flora, as well as frequent opportunistic pathogens of both the upper and lower respiratory tracts. Recently, it has been shown that NTHI can form biofilms both in vitro and in vivo. NTHI strains within in vitro-formed biofilms differentially express both epitopes of lipooligosaccharide (LOS) and the outer membrane proteins P2, P5, and P6, whereas those generated either in a 96-well plate assay in vitro or in a mammalian host have been shown to incorporate a specific glycoform of sialylated LOS within the biofilm matrix. While DNA has been identified as a key component of the biofilm matrix formed in vitro by several bacterial pathogens, here we demonstrate for the first time that in addition to sialylated LOS, the biofilm formed by NTHI in vivo contains both type IV pilin protein and a significant amount of double-stranded DNA. The DNA appeared to be arranged in a dense interlaced meshwork of fine strands as well as in individual thicker “ropes” that span water channels, suggesting that DNA could be imparting structural stability to the biofilm produced by NTHI in vivo. The presence of type IV pilin protein both appearing as small aggregates within the biofilm matrix and tracking along DNA strands supports our observations which showed that type IV pili are expressed by NTHI during experimental otitis media when these bacteria form a biofilm in the middle ear space.

Interaction of high molecular weight kininogen binding proteins on endothelial cells

2004 ◽  
Vol 91 (01) ◽  
pp. 61-70 ◽  
Author(s):  
Baby Tholanikunnel ◽  
Berhane Ghebrehiwet ◽  
Allen Kaplan ◽  
Kusumam Joseph
Keyword(s):  
Molecular Weight ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Surface Proteins ◽  
Factor Xii ◽  
High Molecular Weight Kininogen ◽  
Dot Blot ◽  
Cell Plasma ◽  
Molecular Weight Peak

SummaryCell surface proteins reported to participate in the binding and activation of the plasma kinin-forming cascade includes gC1qR, cytokeratin 1 and u-PAR. Each of these proteins binds high molecular weight kininogen (HK) as well as Factor XII. The studies on the interaction of these proteins, using dot-blot analysis, revealed that cytokeratin 1 binds to both gC1qR and u-PAR while gC1qR and u-PAR do not bind to each other. The binding properties of these proteins were further analyzed by gel filtration. When biotinylated cytokeratin 1 was incubated with either gC1qR or u-PAR and gel filtered, a new, higher molecular weight peak containing biotin was observed indicating complex formation. The protein shift was also similar to the biotin shift. Further, immunoprecipitation of solubilized endothelial cell plasma membrane proteins with anti-gC1qR recovered both gC1qR and cytokeratin 1, but not u-PAR. Immunoprecipitation with anti-u-PAR recovered only u-PAR and cytokeratin 1. By competitive ELISA, gC1qR inhibits u-PAR from binding to cytokeratin 1; u-PAR inhibits gC1qR binding to a lesser extent and requires a 10-fold molar excess. Our data suggest that formation of HK (and Factor XII) binding sites along endothelial cell membranes consists of bimolecular complexes of gC1qR-cytokeratin 1 and u-PAR-cytokeratin 1, with gC1qR binding being favored.

scholarly journals The Type IV Pilin of Burkholderia mallei Is Highly Immunogenic but Fails To Protect against Lethal Aerosol Challenge in a Murine Model

2007 ◽  
Vol 75 (6) ◽  
pp. 3027-3032 ◽  
Author(s):  
Paula J. Fernandes ◽  
Qin Guo ◽  
David M. Waag ◽  
Michael S. Donnenberg
Keyword(s):  
High Titer ◽  
Active Immunization ◽  
Burkholderia Mallei ◽  
Subunit Vaccines ◽  
Passive Protection ◽  
Type Iv ◽  
Type Iv Pilin ◽  
A Subunit ◽  
Pilin Protein

ABSTRACT Burkholderia mallei is the cause of glanders and a proven biological weapon. We identified and purified the type IV pilin protein of this organism to study its potential as a subunit vaccine. We found that purified pilin was highly immunogenic. Furthermore, mice infected via sublethal aerosol challenge developed significant increases in titers of antibody against the pilin, suggesting that it is expressed in vivo. Nevertheless, we found no evidence that high-titer antipilin antisera provided passive protection against a sublethal or lethal aerosol challenge and no evidence of protection afforded by active immunization with purified pilin. These results contrast with the utility of type IV pilin subunit vaccines against other infectious diseases and highlight the need for further efforts to identify protective responses against this pathogen.

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