Unusual Genetic Organization of a Functional Type I Protein Secretion System in Neisseria meningitidis

Abstract

Proteins secreted by Neisseria meningitidis are thought to play important roles in the pathogenesis of meningococcal disease. These proteins include the iron-repressible repeat-in-toxin (RTX) exoprotein FrpC. Related proteins in other pathogens are secreted via a type I secretion system (TOSS), but such a system has not been demonstrated in N. meningitidis. An in silico search of the group B meningococcal genome suggested the presence of a uniquely organized TOSS. Genes encoding homologs of the Escherichia coli HlyB (ATPbinding), HlyD (membrane fusion), and TolC (outer membrane channel) proteins were identified. In contrast to the cistronic organization of the secretion genes in most other rtx operons, the hlyD and tolC genes were adjacent but unlinked to hlyB; neither locus was part of an operon containing genes encoding putative TOSS substrates. Both loci were flanked by genes normally associated with mobile genetic elements. The three genes were shown to be expressed independently. Mutation at either locus resulted in an inability to secrete FrpC and a related protein, here called FrpC2. Successful complementation of these mutations at an ectopic site confirmed the observed phenotypes were caused by loss of function of the putative TOSS genes. We show that genes scattered in the meningococcal genome encode a functional TOSS required for secretion of the meningococcal RTX proteins.During invasive meningococcal disease large amounts of lipooligosaccharide are released from the bacterial surface leading to massive inflammation and pathology (7). The contribution of secreted proteins to meningococcal pathogenesis, however, is not well understood. Exoproteins play a major role in the pathogenesis of many gram-negative pathogens and we have recently demonstrated that meningococcal secreted proteins (MSPs) play a central role in modulation of host cell genes with a role in inflammation and apoptosis (28). Proteins may be secreted by gram-negative bacteria by one of a number of known pathways, including those listed as types I to V (19). We and others have shown that meningococci secrete a number of proteins of a class called autotransporters, which are secreted via the type V pathway. These include the putative virulence factors immunoglobulin A (IgA) protease (22), the adhesin App (13, 30), AutA (1), and the serine protease AspA (NalP) (35,36). Meningococci also have genes with homology to fhaB and fhaC of Bordetella pertussis (21). In this pathogen the major virulence factor filamentous hemagglutinin (product of fhaB) is secreted via the two-partner secretion pathway (a variant of the type V pathway) with the help of the fhaC gene product (15). Meningococci are also known to secrete proteins with homology to the repeat-in-toxin (RTX) family of cytotoxins (33, 34). In other gram-negative pathogens such toxins are secreted via a type I secretion system (TOSS) (5), and the recombinant product of one of the meningococcal genes was shown to be secreted via the Escherichia coli hemolysin TOSS (32). The presence of a TOS...

The TolC-Like Protein of Neisseria meningitidis Is Required for Extracellular Production of the Repeats-in-Toxin Toxin FrpC but Not for Resistance to Antimicrobials Recognized by the Mtr Efflux Pump System

“…In agreement with the reports by Klee et al (9) and Wooldridge et al (32), we identified a 1.403-kb locus in the annotated genome sequence of meningococcal strains MC58 and Z2491 that contains an ORF that would encode a TolC-like protein (data not presented). Analysis of the genome sequence flanking this ORF also revealed a closely linked ORF upstream that encodes a protein similar to the HlyD protein of E. coli.…”

Comparative genomics of Neisseria meningitidis: core genome, islands of horizontal transfer and pathogen-specific genes

“…In Moraxella species and N. meningitidis, other type I secretion systems have been shown to be involved in secretion of RTX toxin-like exoproteins (Angelos et al, 2003;Wooldridge et al, 2005). In N. meningitidis, the function of these RTX toxin-like exoproteins has remained elusive, but in Moraxella bovis and other c-proteobacteria, they appear to be functional haemolysins (Angelos et al, 2003;Wooldridge et al, 2005). The MFP and ABC components of the type I secretion system are often genetically linked to the exported substrate/toxin.…”

“…A third large open reading frame in the transposon (NMC_0891 and a conserved hypothetical protein with significant matches only to N. meningitidis strains) should be examined as a potential substrate for this type I secretion system. The third component necessary for a functional secretion system (TolC) has been identified in N. meningitidis strains (Wooldridge et al, 2005) and is typically not genetically linked to the MFP and ABC components.…”

MisR/MisS Two-Component Regulon in Neisseria meningitidis

“…The last group of genes identified encodes putative cell surface-localized proteins that include several transporters (e.g., the capsular polysaccharide transport protein CtrD) and a putative hemolysin. In particular, the HlyB and HlyD proteins together with TolC, an outer-membrane channel protein that is encoded in the same putative operon as hlyD, constitute the type I secretion machinery responsible for the secretion of several iron-inducible repeat-in-toxin (RTX) toxin homologues (53)(54)(55)63).…”