evaluation-of-host-pathogen-responses-and-vaccine-efficacy-in-mice

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

The Ohio State University

AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the β-sheet for DNA binding, while the α-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a β-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ β-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection.

AmrZ beta-sheet residues are essential for DNA binding and transcriptional control of Pseudomonas aeruginosa virulence genes.

Bordetella pertussis is the causative agent of pertussis, a highly contagious disease of the human respiratory tract. Despite very high vaccine coverage, pertussis has reemerged as a serious threat in the United States and many developing countries. Thus, it is important to pursue research to discover unknown pathogenic mechanisms of B. pertussis. We have investigated a previously uncharacterized locus in B. pertussis, the dra locus, which is homologous to the dlt operons of Gram-positive bacteria. The absence of the dra locus resulted in increased sensitivity to the killing action of antimicrobial peptides (AMPs) and human phagocytes. Compared to the wild-type cells, the mutant cells bound higher levels of cationic proteins and peptides, suggesting that dra contributes to AMP resistance by decreasing the electronegativity of the cell surface. The presence of dra led to the incorporation of d-alanine into an outer membrane component that is susceptible to proteinase K cleavage. We conclude that dra encodes a virulence-associated determinant and contributes to the immune resistance of B. pertussis. With these findings, we have identified a new mechanism of surface modification in B. pertussis which may also be relevant in other Gram-negative pathogens. 

D-alanine modification of a protease-susceptible outer membrane component by the Bordetella pertussis dra locus promotes resistance to antimicrobial peptides and polymorphonuclear leukocyte-mediated killing.

Pertussis, or whooping cough, caused by the obligate human pathogen  is undergoing a worldwide resurgence. The majority of studies of this pathogen are conducted with laboratory-adapted strains which may not be representative of the species as a whole. Biofilm formation by  plays an important role in pathogenesis. We conducted a side-by-side comparison of the biofilm-forming abilities of the prototype laboratory strains and the currently circulating isolates from two countries with different vaccination programs. Compared to the reference strain, all strains examined herein formed biofilms at high levels. Biofilm structural analyses revealed country-specific differences, with strains from the United States forming more structured biofilms. Bacterial hyperaggregation and reciprocal expression of biofilm-promoting and -inhibitory factors were observed in clinical isolates. An association of increased biofilm formation with augmented epithelial cell adhesion and higher levels of bacterial colonization in the mouse nose and trachea was detected. To our knowledge, this work links for the first time increased biofilm formation in bacteria with a colonization advantage in an animal model. We propose that the enhanced biofilm-forming capacity of currently circulating strains contributes to their persistence, transmission, and continued circulation.

Hyperbiofilm Formation by Bordetella pertussis Strains Correlates with Enhanced Virulence Traits.

The reemergence of pertussis or whooping cough in several countries highlights the need for better vaccines. Acellular pertussis vaccines (aPV) contain alum as the adjuvant and elicit Th2 biased immune responses that are less effective in protecting against infection than the reactogenic whole cell pertussis vaccines (wPV), which elicit primarily a Th1/Th17 response. An important goal for the field is to devise aPVs that will induce immune responses similar to wPV. We show that Bordetella Colonization Factor A (BcfA), an outer membrane protein from  has strong adjuvant function and elicits cellular and humoral immune responses to heterologous and  antigens. Addition of BcfA to a commercial aPV resulted in greater reduction of  numbers from the lungs than elicited by aPV alone. More efficient pathogen clearance was accompanied by increased IL-17 and reduced IL-5, and an increased ratio of IgG2/IgG1 antibodies. Thus our results suggest that BcfA improves aPV induced responses by modifying the alum-induced Th2-biased aPV response towards Th1/Th17. A re-designed aPV containing BcfA may allow better control of pertussis reemergence by reshaping immune responses to resemble those elicited by wPV immunization.

The adjuvant Bordetella Colonization Factor A attenuates alum-induced Th2 responses and enhances  clearance from mouse lungs.

Many of the pathogenic species of the genus  have an absolute requirement for nicotinic acid (NA) for laboratory growth. These Gram-negative bacteria also harbor a gene cluster homologous to the  cluster of  which is involved in the aerobic degradation of NA and its transcriptional control. We report here that BpsR, a negative regulator of biofilm formation and Bps polysaccharide production, controls the growth of  by repressing the expression of  genes. The severe growth defect of the Δ strain in Stainer-Scholte medium was restored by supplementation with NA, which also functioned as an inducer of  genes at low micromolar concentrations that are usually present in animals and humans. Purified BpsR protein bound to the  promoter region, and its DNA binding activity was inhibited by 6-hydroxynicotinic acid (6-HNA), the first metabolite of the NA degradative pathway. Reporter assays with the isogenic mutant derivative of the wild-type (WT) strain harboring deletion in , which encodes a putative nicotinic acid hydroxylase responsible for conversion of NA to 6-HNA, showed that 6-HNA is the actual inducer of the  genes in the bacterial cell. Gene expression profiling further showed that BpsR dually activated and repressed the expression of genes associated with pathogenesis, transcriptional regulation, metabolism, and other cellular processes. We discuss the implications of these findings with respect to the selection of pyridines such as NA and quinolinic acid for optimum bacterial growth depending on the ecological niche. BpsR, the previously described regulator of biofilm formation and Bps polysaccharide production, controls  growth by regulating the expression of genes involved in the degradation of nicotinic acid (NA). 6-Hydroxynicotinic acid (6-HNA), the first metabolite of the NA degradation pathway prevented BpsR from binding to DNA and was the actual  inducer. We hypothesize that BpsR enables  bacteria to efficiently and selectively utilize NA for their survival depending on the environment in which they reside. The results reported herein lay the foundation for future investigations of how BpsR and the alteration of its activity by NA orchestrate the control of  growth, metabolism, biofilm formation, and pathogenesis.

The Transcriptional Regulator BpsR Controls the Growth of Bordetella bronchiseptica by Repressing Genes Involved in Nicotinic Acid Degradation.

Poly-β(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.

Article	Year
AmrZ beta-sheet residues are essential for DNA binding and transcriptional control of Pseudomonas aeruginosa virulence genes. Journal of bacteriology\| PubMed ID: 20709902	2010
D-alanine modification of a protease-susceptible outer membrane component by the Bordetella pertussis dra locus promotes resistance to antimicrobial peptides and polymorphonuclear leukocyte-... Journal of bacteriology\| PubMed ID: 24013634	2013
Hyperbiofilm Formation by Bordetella pertussis Strains Correlates with Enhanced Virulence Traits. Infection and immunity\| PubMed ID: 28893915	2017
The adjuvant Bordetella Colonization Factor A attenuates alum-induced Th2 responses and enhances clearance from mouse lungs. Infection and immunity\| PubMed ID: 29531137	2018
The Transcriptional Regulator BpsR Controls the Growth of Bordetella bronchiseptica by Repressing Genes Involved in Nicotinic Acid Degradation. Journal of bacteriology\| PubMed ID: 29581411	2018
PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms. PLoS pathogens\| PubMed ID: 29684093	2018

Rajendar Deora

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