Role of HrpA in Biofilm Formation of Neisseria meningitidis and Regulation of the hrpBAS Transcripts

Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA.
Infection and immunity (Impact Factor: 3.73). 04/2009; 77(6):2285-93. DOI: 10.1128/IAI.01502-08
Source: PubMed


Two-partner secretion systems of gram-negative organisms are utilized in adherence, invasion, and biofilm formation. The HrpAB
proteins of Neisseria meningitidis are members of a two-partner secretion system, and HrpA is established as being important to adherence and intracellular
escape. This study set out to determine the expression pattern of members of the hrpBAS putative operon and to find a functional role for the HrpA protein. The upregulation of these genes was found in situations
of anaerobiosis and cell contact. These observations prompted the study of the function of HrpA in biofilms on human bronchial
epithelial cells. HrpA mutants in encapsulated and unencapsulated NMB strains demonstrated biofilm growth equivalent to that
of the wild-type strain at 6 h but a decreased ability to form biofilms at 48 h. Biofilms formed by hrpA mutants for 48 h on collagen-coated coverslips demonstrated significant reductions compared to those of wild-type strains.
Taken together, these observations imply a role for HrpA in the biofilm structure. Further analysis demonstrated the presence
of HrpA on the surface of the bacterium.

Full-text preview

Available from:
  • Source
    • "Finally, we analyzed strains from another hyperinvasive cc, i.e. cc8, using the same strategy. The TPS island of cc8 strain NMB was recently reported to have a similar organization as that in FAM18 [18]. We sequenced tpsA of another cc8 strain, strain 2996 (Genbank: HQ420264), and found 99% identity at the nucleotide level with tpsA of FAM18. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Two-partner secretion systems in Gram-negative bacteria consist of an outer membrane protein TpsB that mediates the secretion of a cognate TpsA protein into the extracellular milieu. TpsA proteins have diverse, often virulence-related functions, and some of them inhibit the growth of related bacteria. In Neisseria meningitidis, several functions have been attributed to the TpsA proteins. Downstream of the tpsB and tpsA genes, several shorter tpsA-related gene cassettes, called tpsC, are located interspersed with intervening open-reading frames (IORFs). It has been suggested that the tpsC cassettes may recombine with the tpsA gene as a mechanism of antigenic variation. Here, we investigated (i) whether TpsA of N. meningitidis also has growth-inhibitory properties, (ii) whether tpsC cassettes recombine with the tpsA gene, and (iii) what the consequences of such recombination events might be. We demonstrate that meningococcal TpsA has growth-inhibitory properties and that the IORF located immediately downstream of tpsA confers immunity to the producing strain. Although bioinformatics analysis suggests that recombination between tpsC cassettes and tpsA occurs, detailed analysis of the tpsA gene in a large collection of disease isolates of three clonal complexes revealed that the frequency is very low and cannot be a mechanism of antigenic variation. However, recombination affected growth inhibition. In vitro experiments revealed that recombination can be mediated through acquirement of tpsC cassettes from the environment and it identified the regions involved in the recombination. Meningococcal TpsA has growth-inhibitory properties. Recombination between tpsA and tpsC cassettes occurs in vivo but is rare and has consequences for growth inhibition. A recombination model is proposed and we propose that the main goal of recombination is the collection of new IORFs for protection against a variety of TpsA proteins.
    Full-text · Article · Sep 2013 · BMC Genomics
  • Source
    • "Meningococci have been shown to form biofilms also in continuous flow systems on abiotic and biotic surfaces (Lappann et al., 2006; Neil et al., 2009). Such systems assure the continuous supply of nutrients and maintain shear forces that may mimic natural dynamic conditions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Neisseria meningitidis is a common and usually harmless inhabitant of the mucosa of the human nasopharynx, which, in rare cases, can cross the epithelial barrier and cause meningitis and sepsis. Biofilm formation favours the colonization of the host and the subsequent carrier state. Two different strategies of biofilm formation, either dependent or independent on extracellular DNA (eDNA), have been described for meningococcal strains. Here, we demonstrate that the autotransporter protease NalP, the expression of which is phase variable, affects eDNA-dependent biofilm formation in N. meningitidis. The effect of NalP was found in biofilm formation under static and flow conditions and was dependent on its protease activity. Cleavage of the heparin-binding antigen NhbA and the α-peptide of IgA protease, resulting in the release of positively charged polypeptides from the cell surface, was responsible for the reduction in biofilm formation when NalP is expressed. Both NhbA and the α-peptide of IgA protease were shown to bind DNA. We conclude that NhbA and the α-peptide of IgA protease are implicated in biofilm formation by binding eDNA and that NalP is an important regulator of this process through the proteolysis of these surface-exposed proteins.
    Full-text · Article · Nov 2012 · Molecular Microbiology
  • Source
    • "E. coli strains were transformed by chemical competence (Top 10) or by electroporation (DH5α) with a GenePulser (Bio-Rad) according to the manufacturer's protocol. Antibiotic concentrations (μg/ml) for N. meningitidis were: kanamycin 80, chloramphenicol, 5 (Neil & Apicella, 2009), tetracycline, 5, spectinomycin, 60 and erythromycin 3; and for E. coli strains were: ampicillin 100, kanamycin 50, chloramphenicol 34, spectinomycin 100, and erythromycin 300. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Proper periplasmic disulfide bond formation is important for folding and stability of many secreted and membrane proteins, and is catalysed by three DsbA oxidoreductases in Neisseria meningitidis. DsbD provides reducing power to DsbC that shuffles incorrect disulfide bond in misfolded proteins as well as to the periplasmic enzymes that reduce apo-cytochrome c (CcsX) or repair oxidative protein damages (MrsAB). The expression of dsbD, but not other dsb genes, is positively regulated by the MisR/S two-component system. Quantitative real-time PCR analyses showed significantly reduced dsbD expression in all misR/S mutants, which was rescued by genetic complementation. The direct and specific interaction of MisR with the upstream region of the dsbD promoter was demonstrated by electrophoretic mobility shift assay, and the MisR binding sequences were mapped. Further, the expression of dsbD was found to be induced by dithiothrietol (DTT), through the MisR/S regulatory system. Surprisingly, we revealed that inactivation of dsbD can only be achieved in a strain carrying an ectopically located dsbD, in the dsbA1A2 double mutant or in the dsbA1A2A3 triple mutant, thus DsbD is indispensable for DsbA-catalysed oxidative protein folding in N. meningitidis. The defects of the meningococcal dsbA1A2 mutant in transformation and resistance to oxidative stress were more severe in the absence of dsbD.
    Full-text · Article · Mar 2011 · Molecular Microbiology
Show more

Similar Publications