Karl E. Klose

MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival

Francisella tularensis is able to survive and grow within macrophages, a trait that contributes to pathogenesis. Several genes have been identified that are important for intramacrophage survival, including mglA and iglC. F. tularensis is also able to survive within amoebae. It is shown here that F. tularensis mglA and iglC mutant strains are not only defective for survival and replication within the macrophage-like cell line J774, but also within Acanthamoebae castellanii. Moreover, these strains are highly attenuated for virulence in mice, suggesting that a common mechanism underlies intramacrophage and intraamoebae survival and virulence. A 2D gel analysis of cell extracts of wild-type and mglA mutant strains revealed that at least seven prominent proteins were at low levels in the mglA mutant, and one MglA-regulated protein was identified as the IglC protein. RT-PCR analysis demonstrated reduced transcription of iglC and several other known and suspected virulence genes in the mglA mutant. Thus, MglA regulates the transcription of virulence factors of F. tularensis that contribute to intramacrophage and intraamoebae survival.

The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139

Throughout most of history, epidemic and pandemic cholera was caused by Vibrio cholerae of the serogroup O1. In 1992, however, a V. cholerae strain of the serogroup O139 emerged as a new agent of epidemic cholera. Interestingly, V. cholerae O139 forms biofilms on abiotic surfaces more rapidly than V. cholerae O1 biotype El Tor, perhaps because regulation of exopolysaccharide synthesis in V. cholerae O139 differs from that in O1 El Tor. Here, we show that all flagellar mutants of V. cholerae O139 have a rugose colony morphology that is dependent on the vps genes. This suggests that the absence of the flagellar structure constitutes a signal to increase exopolysaccharide synthesis. Furthermore, although exopolysaccharide production is required for the development of a three‐dimensional biofilm, inappropriate exopolysaccharide production leads to inefficient colonization of the infant mouse intestinal epithelium by flagellar mutants. Thus, precise regulation of exopolysaccharide synthesis is an important factor in the survival of V. cholerae O139 in both aquatic environments and the mammalian intestine.

The novel σ54‐ and σ28‐dependent flagellar gene transcription hierarchy of Vibrio cholerae

The human pathogen Vibrio cholerae is a highly motile organism by virtue of a polar flagellum. Flagellar transcriptional regulatory factors have been demonstrated to contribute to V. cholerae virulence, but the role these factors play in the transcription hierarchy controlling flagellar synthesis has been unclear. The flagellar genes revealed by the V. cholerae genome sequence are located in three large clusters, with the exception of the motor genes, which are found in three additional locations. It had previously been demonstrated that the alternative sigma factor σ54 and the σ54‐dependent activators FlrA and FlrC are necessary for flagellar synthesis. The V. cholerae genome sequence revealed the presence of a fliA gene, which is predicted to encode the alternative flagellar sigma factor σ28. A V. choleraeΔfliA mutant strain is non‐motile, and synthesizes a truncated flagellum. Vibrio cholerae FliA complements both V. cholerae and Salmonella typhimurium fliA mutants for motility, consistent with its function as an alternative flagellar sigma factor. Analysis of lacZ transcriptional fusions of the V. cholerae flagellar promoters in both V. cholerae and S. typhimurium identified σ28‐, σ54‐, FlrA‐ and FlrC‐dependent promoters, as well as promoters that were independent of all these factors. Our results support a model of V. cholerae flagellar gene transcription as a novel hierarchy composed of four classes of genes. Class I is composed solely of the gene encoding the σ54‐dependent activator FlrA, which along with the σ54‐holoenzyme form of RNA polymerase activates expression of Class II genes. These genes include structural components of the MS ring, switch and export apparatus, as well as the genes encoding both FliA and FlrC. FlrC, along with σ54‐holoenzyme, activates expression of Class III genes, which include basal body, hook and filament genes. Finally, σ28‐holoenzyme activates expression of Class IV genes, which include additional filament genes as well as motor genes. Thus, this novel V. cholerae flagellar hierarchy has incorporated elements from both the σ54‐dependent Caulobacter crescentus polar flagellar hierarchy and the σ28‐dependent S. typhimurium peritrichous flagellar hierarchy.

Regulation of virulence in Vibrio cholerae: the ToxR regulon

Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera. This disease consists of enormous fluid loss through stools, which can be fatal. Cholera epidemics appear in explosive outbreaks that have occurred repeatedly throughout history. The virulence factors toxin coregulated pilus (TCP) and cholera toxin (CT) are essential for colonization of the host and enterotoxicity, respectively. These virulence factors are under the control of ToxT, an AraC/XylS family protein that activates transcription of the genes encoding TCP and CT. ToxT is under the control of a virulence regulatory cascade known as the ToxR regulon, which responds to environmental stimuli to ensure maximal virulence-factor induction within the human intestine. An understanding of this intricate signaling pathway is essential for the development of methods to treat and prevent this devastating disease.

Allelic exchange in Francisella tularensis using PCR products

We describe here a technique for allelic exchange in Francisella tularensis subsp. novicida utilizing polymerase chain reaction (PCR) products. Linear PCR fragments containing gene deletions with an erythromycin resistance cassette insertion were transformed into F. tularensis. The subsequent ErmR progeny were found to have undergone allelic exchange at the correct location in the genome; the minimum flanking homology necessary was 500 bp. This technique was used to create mglA, iglC, bla, and tul4 mutants in F. tularensis subsp. novicida strains. The mglA and iglC mutants were defective for intramacrophage growth, and the tul4 mutant lacked detectable Tul4 by Western immunoblot, as expected. Interestingly, the bla mutant maintained resistance to ampicillin, indicating the presence of multiple ampicillin resistance genes in F. tularensis.

Phosphorylation of the flagellar regulatory protein FlrC is necessary for Vibrio cholerae motility and enhanced colonization

The human pathogen Vibrio cholerae specifically expresses virulence factors within the host, including cholera toxin (CT) and the toxin co‐regulated pilus (TCP), which allow it to colonize the intestine and cause disease. V. cholerae is a highly motile organism by virtue of a polar flagellum, and motility has been inferred to be an important aspect of virulence, yet the exact role of motility in pathogenesis has remained undefined. The two‐component regulatory system FlrB/FlrC is required for polar flagellar synthesis; FlrC is a σ54‐dependent transcriptional activator. We demonstrate that the transcriptional activity of FlrC affects both motility and colonization of V. cholerae. In a purified in vitro reaction, FlrB transfers phosphate to the wild‐type FlrC protein, but not to a mutant form in which the aspartate residue at amino acid position 54 has been changed to alanine (D54A), consistent with this being the site of phosphorylation of FlrC. The wild‐type FlrC protein, but not the D54A protein, activates σ54‐dependent transcription in a heterologous system, demonstrating that phospho‐FlrC is the transcriptionally active form. A V. cholerae strain containing a chromosomal flrCD54A allele did not synthesize a flagellum and had no detectable levels of transcription of the critical σ54‐dependent flagellin gene flaA. The V. cholerae flrCD54A mutant strain was also defective in its ability to colonize the infant mouse small intestine, approximately 50‐fold worse than an isogenic wild‐type strain. Another mutation of FlrC (methionine 114 to isoleucine; M114I) confers constitutive transcriptional activity in the absence of phosphorylation, but a V. cholerae flrCM114I mutant strain, although flagellated and motile, was also defective in its ability to colonize. The strains carrying D54A or M114I mutant FlrC proteins expressed normal levels of CT and TCP under in vitro inducing conditions. Our results show that FlrC ‘locked’ into either an inactive (D54A) or an active (M114I) state results in colonization defects, thereby demonstrating a requirement for modulation of FlrC activity during V. cholerae pathogenesis. Thus, the σ54‐dependent transcriptional activity of the flagellar regulatory protein FlrC contributes not only to motility, but also to colonization of V. cholerae.

Zebrafish‐Mycobacterium marinum model for mycobacterial pathogenesis

We report here the development of a pathogenesis model utilizing Mycobacterium marinum infection of zebrafish (Danio rerio) for the study of mycobacterial disease. The zebrafish model mimics certain aspects of human tuberculosis, such as the formation of granuloma-like lesions and the ability to establish either an acute or a chronic infection based upon inoculum. This model allows the genetics of mycobacterial disease to be studied in both pathogen and host.

The suckling mouse model of cholera

Vibrio cholerae colonization of the suckling mouse intestine is a commonly used animal model for the human diarrheal disease cholera. This model has a number of advantages as well as disadvantages, and has been extremely useful in the identification and characterization of proven and putative virulence factors involved in human cholera.

Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis

Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathogens. However, a comparative genome analysis of Francisella tularensis allowed us to predict the existence of an alternative route of NAD synthesis in this category A priority pathogen, the causative agent of tularaemia. In this route, the amidation of NaMN to nicotinamide mononucleotide (NMN) occurs before the adenylylation reaction, which converts this alternative intermediate to the NAD cofactor. The first step is catalyzed by NMN synthetase, which was identified and characterized in this study. A crystal structure of this enzyme, a divergent member of the NadE family, was solved at 1.9-Å resolution in complex with reaction products, providing a rationale for its unusual substrate preference for NaMN over NaAD. The second step is performed by NMN adenylyltransferase of the NadM family. Here, we report validation of the predicted route (NaMN → NMN → NAD) in F. tularensis including mathematical modeling, in vitro reconstitution, and in vivo metabolite analysis in comparison with a canonical route (NaMN → NaAD → NAD) of NAD biosynthesis as represented by another deadly bacterial pathogen, Bacillus anthracis.

Regulation of virulence in Vibrio cholerae

Vibrio cholerae causes the diarrheal disease cholera primarily because it expresses a colonization factor (toxin-coregulated pilus; TCP) and a potent toxin (cholera toxin; CT) within the human intestine. While the true environmental signals that induce CT and TCP expression within the intestine remain unknown, much progress has been made identifying the regulatory factors that modulate their expression. Transcriptional regulation of the genes encoding TCP and CT involves a cascade consisting of a number of regulatory factors located on recently acquired mobile genetic elements as well as others residing within the ancestral Vibrio genome. In vivo studies have revealed interesting differences between the regulation of TCP and CT expression in the laboratory and within the intestine.