Bordetella bronchiseptica diguanylate cyclase bdca regulates motility and is important for the establishment of respiratory infection in mice

Bacteria can be motile and planktonic or, alteRNAtively, sessile and participating in the biofilm mode of growth. The transition between these lifestyles can be regulated by a second messenger, cyclic dimeric GMP (c-di-GMP). High intracellular c-di-GMP concentration correlates with biofilm formation and motility inhibition in most bacteria, including Bordetella bronchiseptica, which causes respiratory tract infections in mammals and forms biofilms in infected mice. We previously described the diguanylate cyclase BdcA as involved in c-di-GMP synthesis and motility regulation in B. bronchiseptica; here, we further describe the mechanism whereby BdcA is able to regulate motility and biofilm formation. Amino acid replacement of GGDEF with GGAAF in BdcA is consistent with the conclusion that diguanylate cyclase activity is necessary for biofilm formation and motility regulation, although we were unable to confirm the stability of the mutant protein. In the absence of the bdcA gene, B. bronchiseptica showed enhanced motility, strengthening the hypothesis that BdcA regulates motility in B. bronchiseptica. We showed that c-di-GMP-mediated motility inhibition involved regulation of flagellin expression, as high c-di-GMP levels achieved by expressing BdcA significantly reduced the level of flagellin protein. We also demonstrated that protein BB2109 is necessary for BdcA activity, motility inhibition, and biofilm formation. Finally, absence of the bdcA gene affected bacterial infection, implicating BdcA-regulated functions as important for bacterium-host interactions. This work supports the role of c-di-GMP in biofilm formation and motility regulation in B. bronchiseptica, as well as its impact on pathogenesis. IMPORTANCE Pathogenesis of Bordetella spp., like that of a number of other pathogens, involves biofilm formation. Biofilms increase tolerance to biotic and abiotic factors and are proposed as reservoirs of microbes for transmission to other organs (trachea, lungs) or other hosts. Bis-(3=-5=)-cyclic dimeric GMP (c-di-GMP) is a second messenger that regulates transition between biofilm and planktonic lifestyles. In Bordetella bronchiseptica, high c-di-GMP levels inhibit motility and favor biofilm formation. In the present work, we characterized a B. bronchiseptica diguanylate cyclase, BdcA, which regulates motility and biofilm formation and affects the ability of B. bronchiseptica to colonize the murine respiratory tract. These results provide us with a better understanding of how B. bronchiseptica can infect a host.Fil: Belhart, Keila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Gutierrez, María de la Paz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Zacca, Federico Hernán. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Ambrosis, Nicolás Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Gestal, Monica Cartelle. Georgia State University; Estados UnidosFil: Taylor, Dawn. Georgia State University; Estados UnidosFil: Dahlstrom, Kurt M.. Geisel School of Medicine at Dartmouth; Estados UnidosFil: Harvill, Eric T.. Georgia State University; Estados UnidosFil: O Toole, George. Geisel School of Medicine at Dartmouth; Estados UnidosFil: Sisti, Federico Bernardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Fernandez, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant

<i>B</i>. <i>bronchiseptica</i> infection in a mouse model.

<p>Comparison of <i>B</i>. <i>bronchiseptica</i> bacterial burden in the mouse nose (A) and lungs (B) were determined as described in the Materials and Methods after the indicated days post-infection. Bacteria were intranasally inoculated into external nares with an air displacement pipette (5 x 10⁵ CFU in 50 μl). Green dots correspond to CFU from euthanized mice infected with <i>BbΔlapG</i>. * indicates a significant difference compared to the CFU determined in mice infected with wild type strain, p<0.01.</p

<i>B</i>. <i>bronchiseptica</i> LapG and LapD complement their respective <i>P</i>. <i>fluorescen</i>s mutations.

<p>(A) Biofilm formation by the <i>P</i>. <i>fluorescens ΔlapG</i> mutant (<i>PfΔlapG</i>) carrying plasmids expressing the <i>B</i>. <i>bronchiseptica</i> or <i>P</i>. <i>fluorescens lapG</i> genes was determined by the CV biofilm assay. (B) Cell surface levels of LapA as measured by dot blot. Shown is a representative dot blot assay (top) and quantification of the pixel density (n = 6 ± SD; bottom) to assess the level of cell surface LapA of the <i>P</i>. <i>fluorescens</i> strains expressing LapG or LapD, as well as indicated control strains. *, ** or *** indicate significant differences between strains, p<0.05, p<0.01 and p<0.005 respectively. (C) Biofilm formation by the <i>P</i>. <i>fluorescens ΔlapD</i> mutant (<i>PfΔlapD</i>) carrying plasmids expressing the <i>B</i>. <i>bronchiseptica</i> or <i>P</i>. <i>fluorescens lapD</i> genes was determined by the CV biofilm assay. *, ** or *** indicate significant differences between strains, p<0.05, p<0.01 and p<0.005 respectively.</p

<i>B</i>. <i>bronchiseptica</i> LapG cleaves <i>P</i>. <i>fluorescen</i>s LapA and <i>B</i>. <i>bronchiseptica</i> BrtA.

<p>(A) Cleavage of LapA-HA from the surface of <i>P</i>. <i>fluorescens</i> cells with <i>B</i>. <i>bronchiseptica</i> LapG. Western blot with anti-HA antibodies were used to detect LapA-HA of <i>P</i>. <i>fluorescens</i> released into the supernatant fraction for each indicated strain. (B) Cleavage of N-terminal fragment of BrtA by <i>B</i>. <i>bronchiseptica</i> LapG <i>in vitro</i>. Samples correspond to reaction mixture of <i>E</i>. <i>coli</i> lysate expressing indicated protein or empty vector with <i>E</i>. <i>coli</i> lysate expressing the N-terminus of BrtA (N-term BrtA) incubated at room temperature for 1h. Western blot with anti-HA antibodies was used to detect N-term BrtA.</p

LapG regulates biofilm formation by <i>B</i>. <i>bronchiseptica</i>.

<p>(A) Biofilm formation in different NA concentrations by the wild type carrying a vector control (<i>Bb</i>-pEmpty) or a plasmid over-expressing <i>B</i>. <i>bronchiseptica</i> LapG (<i>Bb-lapG</i>). *, indicates a significant difference versus the <i>Bb</i>-pEmpty in same NA concentration, p<0.001. (B) Biofilm formation by the wild-type strain carrying a vector control (<i>Bb</i>-pEmpty), a strain with a deletion of the <i>lapG</i> gene carrying a vector control (<i>BbΔlapG</i>-pEmpty), and the <i>BbΔlapG</i> mutant complemented with a wild-type copy of the <i>B</i>. <i>bronchiseptica lapG</i> gene (<i>BbΔlapG</i>-pLapG). Biofilm formation was assessed using the microtiter plate assays as described in the Materials and Methods. *, indicates a significant difference in the indicated comparisons, p<0.001.</p

SEM images of <i>B</i>. <i>bronchiseptica</i> biofilms.

<p>(A) The strains <i>Bb</i>-pEmpty (left column of panels) or <i>Bb</i>-pLapG (right column of panels) were grown on vertically submerged coverslips in SS medium alone or supplemented with indicated NA concentrations. After 24 h of growth, biofilms formed at the air–liquid interface were visualized by SEM. (B) Indicated strains were grown on vertically submerged coverslips in SS medium alone (left column of panels) or SS supplemented with 2.0 mM NA (right column of panels).</p

<i>B</i>. <i>bronchiseptica</i> LapA domain organization.

<p>Diagram showing the organization of the <i>lapD</i>, <i>lapG</i> and <i>brtA genes</i> in <i>B</i>. <i>bronchiseptica</i> (top) as well as the domain organization of the BrtA protein (bottom). The narrow arrows indicate the location of the primers used in the RT-PCR experiment. The thick, black arrow indicates the specific cleavage site for LapG. The type I secretion signal (T1SS) and T1SS target domains of BrtA are also shown (orange and cyan, respectively), as is the von Willebrand Factor A (vWFA) domain (light green). The repeat regions are indicated by the 8 yellow barrels. Domains found in repeated regions are also indicated: CADG domain (blue), VCBS domain (green) and IDR (<u>i</u>nter<u>d</u>omain <u>r</u>egion, red and yellow). The CADG and VCBS domain names are based on core conserved amino acid.</p