Alteration of surface lipoprotein profiles is a key strategy that the Lyme disease pathogen, Borrelia burgdorferi, has evolved to be maintained within its enzootic cycle between arthropods and mammals. Accumulated evidence indicates that the central regulatory pathway controlling differential gene expression by B. burgdorferi is the RpoN-RpoS pathway (the 54 -S sigma factor cascade). It was previously shown that activation of the RpoN-RpoS pathway is controlled by Rrp2, a two-component response regulator and 54 -dependent transcriptional activator. The role of Rrp2 in the infectious cycle of B. burgdorferi has not been determined heretofore. In this report, we demonstrate that an rrp2 mutant defective in activating 54 -dependent transcription was unable to establish infection in mice, but the rrp2 mutant was capable of surviving within ticks during and after tick feeding. Because the rrp2 mutant was defective in the production of OspC, an outer surface lipoprotein essential for mammalian host infection, we further examined whether the loss of infectivity of the rrp2 mutant was solely due to the inability to produce OspC. While transformation with a shuttle vector carrying ospC under the control of a constitutive flaB promoter restored infection to an ospC mutant in immunodeficient SCID mice, it could not rescue the avirulent phenotype of the rrp2 mutant. These data indicate that, in addition to controlling OspC, Rrp2 controls another factor(s) essential for B. burgdorferi to establish infection in mammals. Furthermore, microarray analyses revealed that 125 and 19 genes were positively and negatively regulated, respectively, by Rrp2, which provides a foundation for future identification of additional Rrp2-dependent virulence determinants in B. burgdorferi.

This study shows that the Vibrio cholerae RTX toxin is secreted by a four-component type I secretion system (TISS) encoded by rtxB, rtxD, rtxE, and tolC. ATP-binding site mutations in both RtxB and RtxE blocked secretion, demonstrating that this atypical TISS requires two transport ATPases that may function as a heterodimer.Vibrio cholerae, the causative agent of the potentially lifethreatening disease cholera, expresses several factors to establish and cause disease in the host, including toxin-coregulated pili and the secreted ADP-ribosylating cholera toxin, which causes the profuse diarrhea that is a hallmark of cholera disease. Other secreted "accessory" toxins that also contribute to disease pathogenesis have been identified, including a zincmetalloprotease, a pore-forming hemolysin/cytotoxin, and the RTX toxin (12). The zinc-metalloprotease and cholera toxin are exported by a type II secretion system encoded by the eps genes (23). Hemolysin export is dependent upon the hlyB gene (2). However, the mechanism of secretion of the RTX toxin has not yet been characterized.At 484 kDa, the V. cholerae RTX toxin is the second largest single-polypeptide toxin known and causes cell rounding and depolymerization of the actin cytoskeleton in a broad range of cell types. Concurrent with actin stress fiber disassembly, actin monomers are covalently cross-linked into dimers, trimers, and higher multimers, which can be visualized by Western blotting (15,20,26).The RTX toxin is a member of the RTX family of bacterial protein toxins, which includes Escherichia coli hemolysin, Bordetella pertussis adenylate cylcase-hemolysin toxin, and Pasteurella haemolytica and Actinobacillus actinomycetemcomitans leukotoxins. These toxins all share common features: posttranslational maturation, a C-terminal calcium-binding domain of acidic glycine-rich nonapeptide repeats (responsible for the RTX [repeats-in-toxin] nomenclature), and export out of the cell by type I secretion systems (TISS) (31). In this study, we examined a putative TISS apparatus encoded by the operon divergently transcribed from the RTX toxin gene rtxA to assess whether this operon is essential for RTX toxin secretion.Strains. The strains used in this study are listed in Table 1. The operon divergently transcribed from the rtxA operon contains three putative TISS genes. Previously characterized TISS consist of three components: a homodimer of an inner membrane transport ATPase, a trimer of a periplasmic linker protein, and a trimer of an outer membrane porin, exemplified by the Escherichia coli hemolysin TISS, consisting of HlyB, HlyD, and TolC, respectively (3). As shown in Fig. 1A, the hly operon of E. coli is arranged in a single operon, hlyCABD. By contrast, the rtx locus of the V. cholerae genome is composed of two operons containing a total of six open reading frames (ORFs) (16). The first ORF in the left-oriented operon (as shown in Fig. 1A) is 360 nucleotides (nt) in length and encodes a 120-amino-acid conserved hypothetical protein. The second ORF is the putati...

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, secretes several "accessory" toxins, including RTX toxin, which causes the cross-linking of the actin cytoskeleton. RTX toxin is exported to the extracellular milieu by an atypical type I secretion system (T1SS), and we previously noted that RTXassociated activity is detectable only in supernatant fluids from log phase cultures. Here, we investigate the mechanisms for regulating RTX toxin activity in supernatant fluids. We find that exported proteases are capable of destroying RTX activity and may therefore play a role in the growth phase regulation of toxin activity. We determined that the absence of RTX toxin in stationary-phase culture supernatant fluids is also due to a lack of toxin secretion and not attributable to solely proteolytic degradation. We ascertained that the T1SS apparatus is regulated at the transcriptional level by growth phase control that is independent of quorum sensing, unlike other virulence factors of V. cholerae. Additionally, in stationary-phase cultures, all RTX toxin activity is associated with bacterial membranes or outer membrane vesicles.Oral ingestion of Vibrio cholerae by human hosts via contaminated food and water can result in cholera, a severe diarrheal disease that can quickly cause death if left untreated (34). The secreted ADP-ribosylating cholera toxin (CT) causes the profuse diarrhea that is a hallmark of cholera disease and is one of the main virulence factors produced by the bacterium. V. cholerae also expresses the toxin-coregulated pilus (TCP), which is essential for sustained colonization of the human small intestine. In addition, hemagglutinin/protease (HAP), hemolysin, and RTX toxin have been identified as secreted "accessory" toxins that also contribute to disease pathogenesis (11).RTX toxin is a very large toxin with a deduced molecular mass of 484 kDa and causes cell rounding and the depolymerization of the actin cytoskeleton in a broad range of cell types. Concurrent with actin stress fiber disassembly, actin monomers are covalently cross-linked into dimers, trimers, and higher multimers which can be visualized by Western blotting (13,25). rtxA, the 13,635-bp gene encoding the RTX toxin, is the largest open reading frame of the V. cholerae genome (17,25). rtxA is found in both clinical and environmental isolates of V. cholerae, but not in O1 classical biotypes (7,10,25).V. cholerae RTX toxin is a member of the RTX (repeat-intoxin) family of toxins (25) that includes Escherichia coli hemolysin (HlyA) and Bordetella pertussis adenylate cyclase-hemolysin toxin. All RTX exoproteins are exported out of the cell by type I secretion systems (T1SS) (41). These secretion systems commonly consist of three components: an inner membrane transport ATPase, a transmembrane linker protein, and an outer membrane porin, exemplified by the E. coli hemolysin T1SS consisting of HlyB, HlyD, and TolC, respectively (1, 19). V. cholerae RTX toxin is secreted by an atypical T1SS that is composed of four comp...

The BBK32 protein binds to host extracellular ligand fibronectin and contributes to the pathogenesis of Borrelia burgdorferi. Here we showed that expression of the BBK32 gene is influenced by multiple environmental factors and that its regulation is governed by the response regulator Rrp2 and RpoN-RpoS ( 54 -S ) sigma cascade in B. burgdorferi.