Bordetella PlrSR regulatory system controls BvgAS activity and virulence in the lower respiratory tract

Bordetella spp. includes Bordetella pertussis, the causal agent of whooping cough. The Bordetella virulence gene (BvgAS) two-component regulatory system (TCS) is considered the “master virulence regulator” in Bordetella, as it controls expression of all known virulence factor-encoding genes. We show here that another TCS, PlrSR, is required for BvgAS activity in the lower respiratory tract (LRT) and for virulence even when BvgAS is rendered constitutively active, suggesting that it controls critical functions for bacterial survival in the LRT independently of BvgAS. Our data introduce a new layer of complexity to a paradigm of Bordetella virulence control that has held for more than 30 y, and they indicate the existence of previously unknown bacterial factors that may serve as vaccine components and therapeutic targets

The contribution of BvgR, RisA, and RisS to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation in Bordetella bronchiseptica

Bordetella bronchiseptica is a highly contagious respiratory bacterial veterinary pathogen. In this study the contribution of the transcriptional regulators BvgR, RisA, RisS, and the phosphorylation of RisA to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation were evaluated. Next Generation Sequencing (RNASeq) was used to differentiate the global gene regulation of both virulence-activated and virulence-repressed genes by each of these factors. The BvgAS system, along with BvgR, RisA, and the phosphorylation of RisA served in cyclic-di-GMP degradation. BvgR and unphosphorylated RisA were found to temporally regulate motility. Additionally, BvgR, RisA, and RisS were found to be required for biofilm formation

In Vivo Gene Essentiality and Metabolism in Bordetella pertussis

Bordetella pertussis is the causative agent of whooping cough, a serious respiratory illness affecting children and adults, associated with prolonged cough and potential mortality. Whooping cough has reemerged in recent years, emphasizing a need for increased knowledge of basic mechanisms of B. pertussis growth and pathogenicity. While previous studies have provided insight into in vitro gene essentiality of this organism, very little is known about in vivo gene essentiality, a critical gap in knowledge, since B. pertussis has no previously identified environmental reservoir and is isolated from human respiratory tract samples. We hypothesize that the metabolic capabilities of B. pertussis are especially tailored to the respiratory tract and that many of the genes involved in B. pertussis metabolism would be required to establish infection in vivo. In this study, we generated a diverse library of transposon mutants and then used it to probe gene essentiality in vivo in a murine model of infection. Using the CON-ARTIST pipeline, 117 genes were identified as conditionally essential at 1 day postinfection, and 169 genes were identified as conditionally essential at 3 days postinfection. Most of the identified genes were associated with metabolism, and we utilized two existing genome-scale metabolic network reconstructions to probe the effects of individual essential genes on biomass synthesis. This analysis suggested a critical role for glucose metabolism and lipooligosaccharide biosynthesis in vivo. This is the first genome-wide evaluation of in vivo gene essentiality in B. pertussis and provides tools for future exploration. IMPORTANCE Our study describes the first in vivo transposon sequencing (Tn-seq) analysis of B. pertussis and identifies genes predicted to be essential for in vivo growth in a murine model of intranasal infection, generating key resources for future investigations into B. pertussis pathogenesis and vaccine design

Whole genome sequencing of phage resistant Bacillus anthracis mutants reveals an essential role for cell surface anchoring protein CsaB in phage AP50c adsorption

BACKGROUND: Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50(R)) exhibit a mucoid colony phenotype and secrete an extracellular matrix. METHODS: Here we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection. RESULTS: Using whole genome sequence data, we mapped the relevant mutations in six AP50(R) strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains. CONCLUSIONS: Taken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in phage adsorption. (The whole genome sequences generated from this study have been submitted to GenBank under SRA project ID: SRA023659.1 and sample IDs: AP50 R1: SRS113675.1, AP50 R2: SRS113676.1, AP50 R3: SRS113728.1, AP50 R4: SRS113733.1, AP50 R6: SRS113734.1, JB220 Parent: SRS150209.1, JB220 Mutant: SRS150211.1)

Improvements to a Markerless Allelic Exchange System for Bacillus anthracis.

A system was previously developed for conducting I-SceI-mediated allelic exchange in Bacillus anthracis. In this system, recombinational loss of a chromosomally-integrated allelic exchange vector is stimulated by creation of a double-stranded break within the vector by the homing endonuclease I-SceI. Although this system is reasonably efficient and represents an improvement in the tools available for allelic exchange in B. anthracis, researchers are nonetheless required to "pick and patch" colonies in order to identify candidate "exchangeants." In the present study, a number of improvements have been made to this system: 1) an improved I-SceI-producing plasmid includes oriT so that both plasmids can now be introduced by conjugation, thus avoiding the need for preparing electro-competent cells of each integration intermediate; 2) antibiotic markers have been changed to allow the use of the system in select agent strains; and 3) both plasmids have been marked with fluorescent proteins, allowing the visualization of plasmid segregation on a plate and obviating the need for "picking and patching." These modifications have made the process easier, faster, and more efficient, allowing for parallel construction of larger numbers of mutant strains. Using this improved system, the genes encoding the tripartite anthrax toxin were deleted singly and in combination from plasmid pXO1 of Sterne strain 34F2. In the course of this study, we determined that DNA transfer to B. anthracis could be accomplished by conjugation directly from a methylation-competent E. coli strain

Routine Markerless Gene Replacement in Bacillus anthracis

An improved genetic tool suitable for routine markerless allelic exchange in Bacillus anthracis has been constructed. Its utility was demonstrated by the introduction of insertions, deletions, and missense mutations on the chromosome and plasmid pXO1 of the Sterne strain of B. anthracis

Development of Phage Lysin LysA2 for Use in Improved Purity Assays for Live Biotherapeutic Products

Live biotherapeutic products (LBPs), commonly referred to as probiotics, are typically preparations of live bacteria, such as Lactobacillus and Bifidobacterium species that are considered normal human commensals. Popular interest in probiotics has been increasing with general health benefits being attributed to their consumption, but there is also growing interest in evaluating such products for treatment of specific diseases. While over-the-counter probiotics are generally viewed as very safe, at least in healthy individuals, it must be remembered that clinical studies to assess these products may be done in individuals whose defenses are compromised, such as through a disease process, immunosuppressive clinical treatment, or an immature or aging immune system. One of the major safety criteria for LBPs used in clinical studies is microbial purity, i.e., the absence of extraneous, undesirable microorganisms. The main goal of this project is to develop recombinant phage lysins as reagents for improved purity assays for LBPs. Phage lysins are hydrolytic enzymes containing a cell binding domain that provides specificity and a catalytic domain responsible for lysis and killing. Our approach is to use recombinant phage lysins to selectively kill target product bacteria, which when used for purity assays will allow for outgrowth of potential contaminants under non-selective conditions, thus allowing an unbiased assessment of the presence of contaminants. To develop our approach, we used LysA2, a phage lysin with reported activity against a broad range of Lactobacillus species. We report the lytic profile of a non-tagged recombinant LysA2 against Lactobacillus strains in our collection. We also present a proof-of-concept experiment, showing that addition of partially purified LysA2 to a culture of Lactobacillus jensenii (L. jensenii) spiked with low numbers of Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus ) effectively eliminates or knocks down L. jensenii, allowing for clear detection of the contaminating strains. With continued identification and characterization of phage lysins, we hope that the use of recombinant phage lysins in purity assays for products containing live microbials may offer additional tools to help advance product development of LBPs