This work was supported by the National Research Council Canada, for the Plan Nacional de I + D (Ministerio de Econom&#237;a y Competitividad, Spain) and for the Generalitat de Catalunya (Centre de Refer&#232;ncia en Biotecnologia).

The schematic representation of the procedure is shown in Figure 1.
1. Bioinformatic identification of flagella glycosylation island (FGIs) in Aeromonas
<ol>
	<li>To identify the PseAc biosynthetic clusters in the Aeromonas genomes, use the tblastn tool from the NCBI database26. First, retrieve orthologous proteins to PseC and PseI of A. piscicola AH-3 (identification codes are OCA61126.1 and OCA61113.1) from databases of sequenced Aeromonas strains, and then use the tblastn tool to search their translated nucleotides sequences. 
	NOTE: The pse genes flank the Aeromonas FGIs, with pseB and pseC located at one end of the cluster and pseI at the other end. The location of the rest of the pse genes can be different from one FGI group to another.</li>
	<li>Given that those polar flagellin genes of many strains are adjacent to the FGIs, use the tblastn from the NCBI database to find orthologous proteins to the polar flagellins FlaA and FlaB of A. piscicola AH-3 (identification codes are OCA61104.1 and OCA61105.1) and the genes that encode them.</li>
	<li>In addition, Aeromonas FGIs involved in the biosynthesis of heteropolysaccharidic glycans (group II)27 contain several genes encoding enzymes involved in the fatty acid synthesis, such as luxC and luxE. Use the tblastn from the NCBI database to find orthologous proteins to LuxC of A. piscicola AH-3 (identification code is OCA61121.1) and the gene that encodes it.</li>
</ol>
2. Generation of null mutants in flagella glycosylation island genes
NOTE: This method of mutagenesis is based on the allelic exchange of polymerase chain reaction (PCR) in-frame deletion products using the suicide vector pDM428 (GenBank: KC795686.1). Replication of pDM4 vector is lambda pir dependent, and the complete allelic exchange is coerced by utilizing the sacB gene located on the vector.
<ol>
	<li>Construction of PCR in-frame deletion products.
	<ol>
		<li>Design two pairs of primers that amplify DNA regions upstream (A and B primers) and downstream (C and D primers) of the selected gene or region to be deleted (Figure 2B).</li>
		<li>Ensure that Primers A and D have more than 600 bp upstream from the start and downstream from the stop, respectively, of the gene or genes to be deleted. These two primers need to include at the 5&#39; end a restriction site for an endonuclease that allows cloning in pDM4. 
		NOTE: The restriction site included at the 5&#39; end of A and D primers should not be the same as sites within AB or CD amplicons.</li>
		<li>Ensure that Primers B and C are within the gene to be deleted or inside the first and last gene, respectively, of the region to be deleted. Ensure that both primers (B and C) are in-frame and located between 5-6 codons inside the gene. Furthermore, ensure that both primers include at the 5&#39; end a 21 bp complementary sequence (Table 1) that allows joining the DNA amplicons AB and CD.</li>
		<li>Grow the selected Aeromonas strain in 5 mL of tryptic soy broth (TSB) overnight (O/N) at 30 &#176;C. Use a commercially available kit for genomic DNA purification and follow the manufacturer&#39;s instructions (Table of Materials). Quantify 2 &#181;L of the purified DNA using a spectrophotometer. 
		NOTE: Use double distilled water as a blank, with the instrument set to record absorbance at 260 nm. Record absorbance with 2 &#181;L of purified DNA 3-5 times and calculate an average absorbance reading. Use the Beer-Lambert law to calculate the DNA concentration, where A = &#400;.c.l. wherein &#34;A&#34; is absorbance, &#34;&#400;&#34; is the molar average extinction coefficient for DNA, &#34;c&#34; is DNA concentration, and &#34;l&#34; is the path length (refer to the manufacturer&#39;s instruction for the path length of each instrument).</li>
		<li>Use 100 ng of purified chromosomal DNA as the template in two sets of asymmetric PCRs with A-B and C-D primers (Table of Materials). Use a 10:1 molar ratio of A:B and D:C primer pairs (Supplementary Material). 
		NOTE: Asymmetric PCRs allow the preferential amplification of one strand of the template more than the other.</li>
		<li>Analyze the PCR products by electrophoresis in a 1% agarose gel. Use TAE (40 mM Tris-acetate, 1 mM EDTA) as gel running buffer and a power setting of 8 V/cm. To visualize DNA, add ethidium bromide (0.5 &#181;g/mL) to the gel and visualize the PCR products using a bio-imaging station (Table of Materials).</li>
		<li>Excise the amplicons from the gel using a scalpel, purify following the manufacturer&#39;s protocol (Table of Materials) and quantify them.</li>
		<li>Join AB and CD amplicons by their 21 bp overlapping sequences in the 3&#39; end of PCR products (Figure 3). Mix 100 ng of each amplicon (AB and CD) in a PCR tube with PCR reagents (pre-AD PCR), but without primers, and extend using the thermocycler program (Supplementary Material). Then, add 100 &#181;M of A and D primers to the reaction (AD PCR) and amplify as a single fragment (Supplementary Material).</li>
		<li>Analyze the PCR product (AD amplicon) by electrophoresis in a 1% agarose gel using the conditions described in step 2.1.6, excise the amplicon from the gel, purify following the manufacturer&#39;s protocol and quantify the amplicon (Table of Materials).</li>
	</ol>
	</li>
	<li>Construction of pDM4 recombinant plasmid with the in-frame deletion.
	<ol>
		<li>Grow an O/N culture of Escherichia coli cc18&#955; pir containing pDM4 in Luria-Bertani (LB) Miller broth (10 mL) with chloramphenicol (25 &#181;g/mL) at 30 &#176;C.</li>
		<li>Spin down the culture at 5,000 x g at 4 &#176;C and suspend the pellet in 600 &#181;L of sterile distilled water. Perform extraction and purification of plasmid pDM4 using a commercially available plasmid purification kit, following the provider&#39;s instructions (Table of Materials).</li>
		<li>Digest 1 &#181;g of pDM4 and 400 ng of AD amplicon for 2 h with an endonuclease able to digest both ends of the AD amplicon and the cloning site of pDM4 (Figure 3). Follow the enzyme manufacturer&#39;s protocol for reaction conditions (Table of Materials).</li>
		<li>Clean up the digested plasmid and amplicon using a DNA purification kit and quantify them following the manufacturer&#39;s instructions (Table of Materials).</li>
		<li>To prevent religation of the linearized pDM4, remove the phosphate group from both 5&#39; ends using the alkaline phosphatase enzyme following the manufacturer&#39;s instructions (Table of Materials). Then, clean up the treated plasmid and quantify it using a spectrophotometer as described in step 2.1.4 (Table of Materials).</li>
		<li>Combine 150 ng of the digested and phosphatase alkaline treated pDM4 with 95-100 ng of digested AD amplicon at a molar vector:insert ratio of 1:4. Prepare the ligation reaction in a volume of 15 &#181;L, following the manufacturer&#39;s instructions (Table of Materials).</li>
		<li>Incubate O/N at 20 &#176;C or over the weekend at 4 &#176;C. After incubation, inactivate the T4 ligase at 70 &#176;C for 20 min.</li>
		<li>Use ligation to introduce the plasmid into Escherichia coli MC1061&#955;pir strain by electroporation. Use electrocompetent bacteria and 2 mm-gap electroporation cuvettes.</li>
	</ol>
	</li>
	<li>Preparation of electrocompetent E. coli and electroporation of pDM4 recombinant plasmid
	<ol>
		<li>Inoculate a single colony of E. coli MC1061&#955;pir strain into Luria-Bertani (LB) Miller broth and grow O/N at 30 &#176;C with 200 rpm shaking.</li>
		<li>Dilute 2 mL of the bacterial culture in 18 mL of LB broth and incubate at 30 &#176;C with shaking (200 rpm) until an optical density (OD600) between 0.4-0.6 is attained.</li>
		<li>Pellet the bacterial culture by centrifugation at 5,000 x g and 4 &#176;C for 15 min. Discard the supernatant and suspend the pellet in 40 mL of chilled distilled H2O.</li>
		<li>Repeat this cleaning step two more times to remove all culture salts. Finally, suspend the pellet in 4 mL of chilled distilled H2O and transfer 1 mL of the suspension to each of four conical 1.5 mL microfuge tubes.</li>
		<li>Pellet the bacterial culture by centrifugation at 14,000 x g and 4 &#176;C for 5 min. Remove the supernatant without disturbing the pellet and suspend each pellet in 100 &#181;L of chilled distilled H2O.</li>
		<li>Add 3-3.5 &#181;L of the ligation mixture to each tube and incubate on ice for 5 min. Then, transfer the contents of each tube to a chilled 2 mm-gap electroporation cuvette and apply 2 kV, 129 &#937;, and time constant around 5 ms. 
		NOTE: Pre-cool the electroporation cuvettes on ice. Maintain the bacteria on ice during the entire procedure.</li>
		<li>After electroporation, add 250 &#181;L of SOC medium to each of the four cuvettes to recover their contents, transfer them to a culture tube (about 1 mL), and incubate for 1 h at 30 &#176;C with shaking (200 rpm).</li>
		<li>Plate the transformed cells on Luria-Bertani (LB) agar plates supplemented with chloramphenicol (25 &#181;g/mL) to ensure all colonies growing in the plate have the plasmid backbone.</li>
		<li>Pick some colonies from the LB agar plates with chloramphenicol and incubate O/N at 30 &#176;C. When colonies grow, check insertion of the deletion construct into pDM4 by PCR using primers that flank the pDM4 cloning side: pDM4for and pDM4rev (Table 1), and lysed colonies as the template (Supplementary Material).</li>
		<li>Pick one colony with a sterile toothpick and dip it into a 1.5 mL tube containing 15 &#181;L of 1% Triton X-100, 20 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0. Incubate the tubes at 100 &#176;C for 5 min, and then 1 min on ice.</li>
		<li>Spin the tubes in a microfuge at 12,000 x g for 10 min to pellet bacteria and debris. Use 1 &#181;L of the supernatant as the template in the PCR reaction (Table of Materials). The annealing temperature of the primer pair is 50 &#176;C, and the PCR reaction requires 10% DMSO (Supplementary Material).</li>
		<li>Inoculate the colonies that amplified the product of interest in 10 mL of LB broth with chloramphenicol (25 &#181;g/mL) and grow O/N at 30 &#176;C. Extract the plasmid from cultures as described in steps 2.2.1-2.2.2. Sequence using the pDM4 primers following the manufacturer&#39;s instructions (Table of Materials).</li>
	</ol>
	</li>
	<li>Transfer of in-frame deletion to the Aeromonas strain 
	NOTE: The pDM4 recombinant plasmid has to be introduced into the selected Aeromonas strain by triparental mating (Figure 4). Aeromonas strain has to be rifampicin-resistant to be selected after the triparental mating. Therefore, grow the selected strains O/N on TSB at 30 &#176;C, centrifuge 2 mL, 4 mL, and 6 mL of cultures at 5,000 x g for&#160;15 min, suspend each pellet in 100 &#181;L of TSB, and plate in TSA with rifampicin (100 &#181;g/mL).
	<ol>
		<li>Prepare O/N cultures of E. coli MC1061&#955;pir with the pDM4 recombinant plasmid on LB agar with 25 &#181;g/mL chloramphenicol (donor strain), the Aeromonas strain rifampicin-resistant on TSA with 100 &#181;g/mL rifampicin (recipient strain), and the E. coli HB101 with the pRK2073 plasmid on LB agar with 80 &#181;g/mL spectinomycin (helper strain) at 30 &#176;C.</li>
		<li>When the colonies have grown, suspend the same number of colonies (10-15 colonies) of the donor, recipient, and helper strains gently with a sterile toothpick in three LB tubes with 150 &#181;L of LB.</li>
		<li>Then, on an LB agar plate, without antibiotics, mix the three bacterial suspensions in two drops at a recipient: helper: donor ratio of 5:1:1 (50 &#181;L of recipient, 10 &#181;L of the donor, and 10 &#181;L of helper). Incubate the plate face up O/N at 30 &#176;C. Perform negative control of conjugation using the donor strain without recombinant plasmid. 
		NOTE: The helper strain carries the conjugative plasmid pRK2073 and assists the transfer of the pDM4 into the Aeromonas. Do not use vortex to suspend the donor, recipient, and helper strains to avoid breaking the pili.</li>
		<li>Harvest bacteria from the LB agar plate by adding 1 mL of LB broth and spreading it with a sterile glass spreader to obtain a bacterial suspension. Use a pipette to transfer the bacterial suspension to a conical 1.5 mL microfuge tube and vortex vigorously.</li>
		<li>To select the recipient colonies containing the pDM4 recombinant plasmid, plate 100 &#181;L of the harvested bacterial suspension on LB agar plates with rifampicin (100 &#181;g/mL) and chloramphenicol (25 &#181;g/mL).</li>
		<li>Spin down 200 &#181;L and 600 &#181;L of the samples in a microfuge at 5,000 x g for&#160;15 min, suspend the pellet in 100 &#181;L of LB broth and plate on LB agar with rifampicin (100 &#181;g/mL) and chloramphenicol (25 &#181;g/mL). Incubate all plates for 24-48 h at 30 &#176;C.</li>
		<li>Pick up the grown colonies, transfer to LB plates with rifampicin (100 &#181;g/mL) and chloramphenicol (25 &#181;g/mL), and incubate them O/N at 30 &#176;C. Then, confirm that the colonies are Aeromonas by the oxidase test29 and that pDM4 recombinant plasmid was inserted (first recombination) into the specific chromosomal region by PCR using the E and F primers (Supplementary Material), which bind outside the region amplified with the A and D primers (Table of Materials). As described in step 2.3.11, use 1 &#181;L of lysed colonies as the template.</li>
		<li>To complete the allelic exchange for the in-frame deletion, coerce the colonies with the integrated suicide plasmid to a second recombination. Grow the recombinant colonies in 2 mL of LB with 10% sucrose and without antibiotics, O/N at 30 &#176;C.</li>
		<li>Plate 100 &#181;L of the bacterial cultures from step 2.4.8 on LB agar plate with sucrose (10%). Also, plate 100 &#181;L of different culture dilutions (10-2-10-4) and incubate O/N at 30 &#176;C.</li>
		<li>To confirm the loss of pDM4, pick up the colonies, transfer to LB plates with and without chloramphenicol (25 &#181;g/mL), incubate them O/N at 30 &#176;C, and then select the chloramphenicol sensitive colonies.</li>
		<li>Analyze the sensitive colonies by PCR using the E-F primers pair and 1 &#181;L of lysed colonies as the template (Table of Materials and Supplementary Material). Select the colonies whose PCR product correlated with the size of the deleted construct.</li>
	</ol>
	</li>
</ol>
3. Motility assays
NOTE: In some bacterial species with glycosylated flagella, modifications in the levels of glycosylation or glycan composition affect the assembly of flagellins, which is usually reflected as a motility reduction or absence of motility. Therefore, two motility assays were performed with the null mutants.
<ol>
	<li>Motility assay in liquid media
	<ol>
		<li>Culture Aeromonas strains O/N in 1 mL of TSB at 25-30 &#176;C. To adhere a microscope cover slide to an excavated slide, pick up a small amount of silicone with the tip of a toothpick and put a small drop on the four corners of the cover slide. Then, deposit 1 &#181;L of the Aeromonas culture in the middle of the cover slide and mix in a drop of fresh TSB media (2 &#181;L).</li>
		<li>Place an excavated slide on the cover slide. Match the excavation with the deposited drop, press gently, and turn the slide upside down.</li>
		<li>Analyze swimming motility by light microscopy using an optic microscope (Table of Materials) with a 100x objective using immersion oil. 
		&#8203;NOTE: The wild-type strain of Aeromonas must be used as the positive control. As a negative control, use a non-flagellated bacterium such as Klebsiella.</li>
	</ol>
	</li>
	<li>Motility assay in semi-solid media
	<ol>
		<li>Culture Aeromonas strains O/N in TSB (1 mL) at 25-30 &#176;C. Then, transfer 3 &#181;L of the culture onto the center of a soft agar plate (1% tryptone, 0.5% NaCl, 0.25% bacto agar) and incubate the plate face up O/N at 25-30 &#176;C.</li>
		<li>Using a ruler, measure the bacterial migration through the agar from the plate&#39;s center toward the periphery.</li>
	</ol>
	</li>
</ol>
4. Flagella purification
<ol>
	<li>Isolation of flagella anchored to the bacterial surface
	<ol>
		<li>Culture Aeromonas strains O/N in TSB (10 mL) at 25-30 &#176;C. Then, expand the culture into a flask with TSB (900 mL) and let it grow O/N at 25-30 &#176;C with shaking (200 rpm).</li>
		<li>Centrifuge at 5,000 x g for 20 min at 4 &#176;C. Suspend the pellet in 20 mL of 100 mM Tris-HCl buffer (pH 7.8).</li>
		<li>To remove the anchored flagella, shear the suspension in a vortex with a glass bar (2-3 mm diameter) for 3-4 min, and then pass repetitively (minimum six times) through a 21-G syringe.</li>
		<li>Pellet bacteria by two consecutive centrifugations at 4 &#176;C: first, at 8,000 x g for 30 min. Remove the supernatant and centrifuge at 18,000 x g for 20 min. Collect the supernatant, which contains free flagella.</li>
		<li>Ultracentrifuge the supernatant at 100,000 x g for 1 h at 4 &#176;C and suspend the pellet in 150 &#181;L of 100 mM Tris-HCl (pH 7.8) plus 2 mM EDTA buffer. The pellet contains flagella filaments.</li>
		<li>Analyze 5-10 &#181;L of the resuspended pellet from step 4.1.5 by electrophoresis in a 12% SDS-Polyacrylamide gel and visualize the proteins using Coomassie blue staining. 
		NOTE: Follow the manufacturer&#39;s instructions for protein electrophoresis and gel staining. As a positive control of glycosylated flagellin, use the purified flagella of the wild-type strain. Purify the enriched fraction of flagella in a cesium chloride (ClCs) gradient.</li>
	</ol>
	</li>
	<li>Cesium chloride gradient to purify flagella.
	<ol>
		<li>Mix 12.1 g of CsCl with 27 mL of distilled H2O.</li>
		<li>Transfer the enriched fraction of flagella (150 &#181;L) to a thin-walled ultra-clear tube (14 mm x 89 mm) (Table of Materials) and fill it with 12 mL of CsCl solution.</li>
		<li>Ultracentrifuge the tube at 60,000 x g for 48 h at 4 &#176;C in a swinging bucket rotor (Table of Materials).</li>
		<li>Collect the flagella band into the CsCl gradient with a syringe and dialyze against 100 mM Tris-HCl (pH 7.8) plus 2 mM EDTA. Then, analyze the dialyzed flagella by electrophoresis in a 12% SDS-Polyacrylamide gel and Coomassie blue staining (step 4.1.6) or by mass spectrometry.</li>
	</ol>
	</li>
</ol>

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M., Tom&#225;s, J. M., Merino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+glycosylation+of+polar+and+lateral+flagellins+in+Aeromonas+hydrophila+AH-3.">Differential glycosylation of polar and lateral flagellins in Aeromonas hydrophila AH-3.</a> Journal of Biological Chemistry. 287 (33), 27851-27862 (2012).</li><li>Canals, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-structural+flagella+genes+affecting+both+polar+and+lateral+flagella-mediated+motility+in+Aeromonas+hydrophila.">Non-structural flagella genes affecting both polar and lateral flagella-mediated motility in Aeromonas hydrophila.</a> Microbiology. 153, 1165-1175 (2007).</li><li>Howard, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Campylobacter+jejuni+glycosylation+island+important+in+cell+charge,+legionaminic+acid+biosynthesis,+and+colonization+of+chickens.">Campylobacter jejuni glycosylation island important in cell charge, legionaminic acid biosynthesis, and colonization of chickens.</a> Infection and Immunity. 77 (6), 2544-2556 (2009).</li><li>Verma, A., Arora, S. K., Kuravi, S. K., Ramphal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+specific+amino+acids+in+the+N-terminus+of+Pseudomonas+aeruginosa+flagellin+and+of+flagellin+glycosylation+in+the+innate+immune+response.">Roles of specific amino acids in the N-terminus of Pseudomonas aeruginosa flagellin and of flagellin glycosylation in the innate immune response.</a> Infection and Immunity. 73 (12), 8237-8246 (2005).</li><li>Lamy, B., Baron, S., Barraud, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aeromonas:+the+multifaceted+middleman+in+the+One+Health+world.">Aeromonas: the multifaceted middleman in the One Health world.</a> Current Opinion in Microbiology. 65, 24-32 (2022).</li><li>Gav&#237;n, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+flagella+of+Aeromonas+species+are+essential+for+epithelial+cell+adherence+and+biofilm+formation.">Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation.</a> Molecular Microbiology. 43 (2), 383-397 (2002).</li><li>Altschul, F. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gapped+BLAST+and+PSI-BLAST:+a+new+generation+of+protein+database+search+programs.">Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.</a> Nucleic Acids Research. 25 (17), 3389-3402 (1997).</li><li>Forn-Cun&#237;, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+flagella+glycosylation+in+Aeromonas:+Genomic+characterization+and+Involvement+of+a+specific+glycosyltransferase+(Fgi-1)+in+heterogeneous+flagella+glycosylation.">Polar flagella glycosylation in Aeromonas: Genomic characterization and Involvement of a specific glycosyltransferase (Fgi-1) in heterogeneous flagella glycosylation.</a> Frontiers in Microbiology. 11, 595697 (2021).</li><li>Milton, D. L., O'Toole, R., Horstedt, P., Wolf-Watz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellin+A+is+essential+for+the+virulence+of+Vibrio+anguillarum.">Flagellin A is essential for the virulence of Vibrio anguillarum.</a> Journal of Bacteriol.ogy. 178 (5), 1310-1319 (1996).</li><li>Kov&#225;cs, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Pseudomonas+pyocyanea+by+the+oxidase+reaction.">Identification of Pseudomonas pyocyanea by the oxidase reaction.</a> Nature. 178 (4535), 703 (1956).</li><li>Fulton, K. M., Mendoza-Barber&#225;, E., Twine, S. M., Tom&#225;s, J. M., Merino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+glycosylated+and+lateral+non-glycosylated+flagella+from+Aeromonas+hydrophila+strain+AH-1+(Serotype+O11).">Polar glycosylated and lateral non-glycosylated flagella from Aeromonas hydrophila strain AH-1 (Serotype O11).</a> International Journal of Molecular Sciences. 16 (12), 28255-28269 (2015).</li><li>Khider, M., Willassen, N. P., Hansen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+alternative+sigma+factor+RpoQ+regulates+colony+morphology,+biofilm+formation+and+motility+in+the+fish+pathogen+Aliivibrio+salmonicida.">The alternative sigma factor RpoQ regulates colony morphology, biofilm formation and motility in the fish pathogen Aliivibrio salmonicida.</a> BMC Microbiology. 18 (1), 116 (2018).</li><li>Pawar, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+genetic+tools+to+tackle+c-di-GMP-dependent+signalling+in+Pseudomonas+aeruginosa.">Novel genetic tools to tackle c-di-GMP-dependent signalling in Pseudomonas aeruginosa.</a> Journal of Applied Microbiology. 120 (1), 205-217 (2016).</li><li>Vander Broek, W., Chalmers, K. J., Stevens, M. P., Stevens, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomic+analysis+of+Burkholderia+pseuomallei+Bsa+Type+III+secretion+system+effectors+using+hypersecreting+mutants.">Quantitative proteomic analysis of Burkholderia pseuomallei Bsa Type III secretion system effectors using hypersecreting mutants.</a> The American Society for Biochemistry and Molecular Biology. 14 (4), 905-916 (2015).</li></ol>

The authors have nothing to disclose.

The critical early step of this method is the identification of regions involved in the glycosylation of flagella and putative GTs because these enzymes show high homology and are involved in many processes. Bioinformatic analysis of Aeromonas genomes in public databases shows that this region is adjacent to the polar flagella region 2, which contains the flagellin genes in many strains and contains genes involved in the biosynthesis of pseudaminic acid27. This has made it possible to dev...

Protein glycosylation has been described in both Gram-positive and Gram-negative bacteria and consists of the covalent attachment of a glycan to an amino acid side chain1,2. In prokaryotes, this process usually occurs via two major enzymatic mechanisms: O- and N-glycosylation3. In O-glycosylation, the glycan is attached to the hydroxyl group of a serine (Ser) or threonine (Thr) residue. In N-glycosylation, the glycan is attached to the side chain amide nitrogen of an asparagine (Asn) residue within the tripeptide sequences Asn-X-Ser/Thr, where X could be any amino acid except proline.
Glycans can adopt linear or branched structures and are composed of monosaccharides or polysaccharides covalently linked by glycosidic bonds. In prokaryotes, glycans usually show diversity in sugar composition and structure in comparison to eukaryotic glycans4. Furthermore, two different bacterial glycosylation pathways that differ in how the glycan is assembled and transferred to the acceptor protein have been described: sequential and en bloc glycosylation5,6. For sequential glycosylation, the complex glycan is built up directly on the protein by successive addition of monosaccharides. In en bloc glycosylation, a pre-assembled glycan is transferred to the protein from a lipid-linked oligosaccharide by a specialized oligosaccharyltransferase (OTase). Both pathways have been shown to be involved in N- and O-glycosylation processes7.
Protein glycosylation has a role in modulating the physicochemical and biological properties of proteins. The presence of a glycan can influence how the protein interacts with its ligand, which affects the biological activity of the protein, but can also affect protein stability, solubility, susceptibility to proteolysis, immunogenicity, and microbe-host interactions8,9. However, several glycosylation parameters, such as the number of glycans, glycan composition, position, and attachment mechanism, could also affect protein function and structure.
Glycosyltransferases (GTs) are the key enzymes in the biosynthesis of complex glycans and glycoconjugates. These enzymes catalyze the glycosidic bond formation between a sugar moiety from an activated donor molecule and a specific substrate acceptor. GTs can use both nucleotides and non-nucleotides as donor molecules and target different substrate acceptors, such as proteins, saccharides, nucleic acids, and lipids10. Therefore, understanding GTs at the molecular level is important to identify their mechanisms of action and specificity, and also enables understanding how sugar composition of glycans that modify relevant molecules are related to pathogenicity. The Carbohydrate Active enzyme database (CAZy)11 classifies GTs according to their sequence homology, which provides a predictive tool since, in most of the GT families, the structural fold and mechanisms of action are invariant. However, four reasons make it difficult to predict substrate specificity of many GTs: 1) no clear sequence motif determining substrate specificity has been determined in prokaryotes12, 2) many GTs and OTases show substrate promiscuity13,14, 3) functional GTs are difficult to produce in high yield in recombinant form and 4) the identification of both donor and acceptor substrates is complex. Despite this, recent mutagenesis studies have made it possible to obtain significant advances in the understanding of catalytic mechanisms and subtract binding of GTs.
In bacteria, O-glycosylation seems to be more prevalent than N-glycosylation. The O-glycosylation sites do not show a consensus sequence, and many of the O-glycosylated proteins are secreted or cell-surface proteins, such as flagellins, pili, or autotransporters1. Flagellin glycosylation shows variability in the number of acceptor sites, glycan composition, and structure. For example, Burkholderia spp flagellins have only one acceptor site, while in Campylobacter jejuni, flagellins have as many as 19 acceptor sites15,16. Furthermore, for some bacteria, the glycan is a single monosaccharide, while other bacteria possess heterogeneous glycans compromised of different monosaccharides to form oligosaccharides. This heterogenicity occurs even among strains of the same species.&#160;Helicobacter flagellins are only modified by pseudaminic acid (PseAc)17, and Campylobacter flagellins can be modified by PseAc, the acetamidino form of the pseudaminic acid (PseAm) or legionaminic acid (LegAm), and glycans derived from these sugars with acetyl, N-acetylglucosamine, or propionic substitutions18,19. In Aeromonas, flagellins are modified by glycans whose composition ranges from a single PseAc acid derivative to a heteropolysaccharide20, and the attachment of glycans to the flagellin monomers is always via a PseAc derivative.
In general, glycosylation of flagellins is essential for flagellar filament assembly, motility, virulence, and host specificity. However, while flagellins of C. jejuni16, H. pylori17, and Aeromonas sp.21 cannot assemble into filament unless the protein monomers are glycosylated, Pseudomonas spp. and Burkholderia spp.15 do not require glycosylation for flagella assembly. Furthermore, in some C. jejuni strains, changes in sugar composition of the flagella glycan affect bacterial-host interaction and may play a role in evading certain immune responses16. Autoagglutination is another phenotypic characteristic affected by modifications in the composition of glycans associated with flagellins. A lower autoagglutination leads to a reduction in the ability to form microcolonies and biofilm22. In some bacteria, the ability of flagella to trigger a pro-inflammatory response was linked to flagellin glycosylation. Thus, in P. aeruginosa, glycosylated flagellin induces a higher pro-inflammatory response than unglycosylated23.
Aeromonas are Gram-negative bacteria ubiquitous in the environment, which allows them to be at the interface of all One Health components24. Mesophilic Aeromonas have a single polar flagellum, which is constitutively produced. More than half of clinical isolates also express lateral flagellin, inducible in high viscosity media or plates. Different studies have related both flagella types with the early stages of bacterial pathogenesis25. While polar flagellins reported to date are O-glycosylated at 5-8 Ser or Thr residues of its central immunogenic domains, lateral flagellins are not O-glycosylated in all the strains. Although polar flagella glycans from different strains show diversity in their carbohydrate composition and chain length20, the linking sugar has been shown to be a pseudaminic acid derivative.
The goal of this manuscript is to describe a method to obtain null mutants in specific GTs or chromosomal regions containing GTs to analyze their involvement in the biosynthesis of relevant polysaccharides and in bacterial pathogenicity, as well as the role of the glycan itself. As an example, we identify and delete a chromosomal region containing GTs of Aeromonas to establish its involvement in polar flagellin glycosylation and analyze the role of the flagellin glycan. We show how to delete a specific GT to establish its function in the biosynthesis of this glycan and the role of modified glycan. Although using Aeromonas as an example, the principle can be used to identify and study flagella glycosylation islands of other Gram-negative bacteria and analyze the function of GTs involved in the biosynthesis of other glycans such as the O-antigen lipopolysaccharide.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ABI PRISM Big Dye Terminator v. 3.1 Cycle Sequencing Ready Reaction Kit</td>
			<td>Applied Biosystems</td>
			<td>4337455</td>
			<td>Used for sequencing</td>
		</tr>
		<tr>
			<td>AccuPrime Taq DNA Polymerase, high fidelity</td>
			<td>Invitrogen</td>
			<td>12346-086</td>
			<td>Used for amplification of AB, CD and AD fragments</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Conda-Pronadise</td>
			<td>8008</td>
			<td>Used for DNA electrophoresis</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase, calf intestinal (CIAP)</td>
			<td>Promega</td>
			<td>M1821</td>
			<td>Used to remove phosphate at the 5&#8217; end</td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>Becton Dickinson</td>
			<td>214010</td>
			<td>Use for motility analysis</td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>Promega</td>
			<td>R6021</td>
			<td>Used for endonuclease restriction</td>
		</tr>
		<tr>
			<td>BglII</td>
			<td>Promega</td>
			<td>R6081</td>
			<td>Used for endonuclease restriction</td>
		</tr>
		<tr>
			<td>BioDoc-It Imagin System</td>
			<td>UVP</td>
			<td></td>
			<td>Bio-imaging station used for DNA visualization</td>
		</tr>
		<tr>
			<td>Biotaq polymerase</td>
			<td>Bioline</td>
			<td>BIO-21040</td>
			<td>Used for colony screening</td>
		</tr>
		<tr>
			<td>Cesium chloride</td>
			<td>Applichem</td>
			<td>A1126,0100</td>
			<td>Used for flagella purification</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Applichem</td>
			<td>A1806,0025</td>
			<td>Used for triparental mating</td>
		</tr>
		<tr>
			<td>Cytiva illustra GFX PCR DNA and Gel Band Purification Kit</td>
			<td>Cytivia</td>
			<td>28-9034-71</td>
			<td>Used for purification of PCR amplicons and DNA fragments.</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Applichem</td>
			<td>131026.1211</td>
			<td>Used for DNA electrophoresis</td>
		</tr>
		<tr>
			<td>Electroporation cuvettes 2 mm gap</td>
			<td>VWR</td>
			<td>732-1133</td>
			<td>Used for transformation</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Applichem</td>
			<td>A1152,0025</td>
			<td>Use for DNA visualization</td>
		</tr>
		<tr>
			<td>HyperLadder 1 Kb marker</td>
			<td>Bioline</td>
			<td>BIO-33053</td>
			<td>DNA marker</td>
		</tr>
		<tr>
			<td>Invitrogen Easy-DNA gDNA Purification Kit</td>
			<td>Invitrogen</td>
			<td>10750204</td>
			<td>Used for bacterial chromosomal DNA purification</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) Miller agar</td>
			<td>Condalab</td>
			<td>996</td>
			<td>Used for Escherichia coli culture</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) Miller broth</td>
			<td>Condalab</td>
			<td>1551</td>
			<td>Used for Escherichia coli culture</td>
		</tr>
		<tr>
			<td>Nanodrop ND-1000</td>
			<td>NanoDrop Techonologies Inc</td>
			<td></td>
			<td>Spectrophotometer used for DNA quantification</td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Applichem</td>
			<td>A2220,0005</td>
			<td>Used for triparental mating</td>
		</tr>
		<tr>
			<td>SOC Medium</td>
			<td>Invitrogen</td>
			<td>15544034</td>
			<td>Used for electroporation recovery</td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Applichem</td>
			<td>A3834,0005</td>
			<td>Used for triparental mating</td>
		</tr>
		<tr>
			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman</td>
			<td>331362</td>
			<td>Used for flagella purification</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Invitrogen</td>
			<td>15224017</td>
			<td>Used for ligation reaction</td>
		</tr>
		<tr>
			<td>Trypticasein soy agar</td>
			<td>Condalab</td>
			<td>1068</td>
			<td>Used for Aeromonas grown</td>
		</tr>
		<tr>
			<td>Trypticasein soy broth</td>
			<td>Condalab</td>
			<td>1224</td>
			<td>Used for Aeromonas grown</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Condalab</td>
			<td>1612</td>
			<td>Use for motility analysis</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Applichem</td>
			<td>A2264,0500</td>
			<td>Used for DNA electrophoresis and flagella purification</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Applichem</td>
			<td>A4975,0100</td>
			<td>Used for bacterial lysis</td>
		</tr>
		<tr>
			<td>Ultra Clear tubes (14 mm x 89 mm)</td>
			<td>Beckman</td>
			<td>344059</td>
			<td>Used for flagella purification</td>
		</tr>
		<tr>
			<td>Veriti 96 well Thermal Cycler</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td>Used for PCR reactions</td>
		</tr>
		<tr>
			<td>Zyppy Plasmid Miniprep II Kit</td>
			<td>Zymmo research</td>
			<td>D4020</td>
			<td>Used for isolation of plasmid DNA</td>
		</tr>
	</tbody>
</table>

generation of null mutants to elucidate the role of bacterial glycosyltransferases in bacterial motility

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

This methodology provides an effective system to generate null mutants in genes or chromosomal regions of Aeromonas that can affect flagella glycosylation and the role of flagella filament (Figure 1).
The protocol starts with the bioinformatic identification of putative FGIs and the genes encoding GTs presents in this region. In Aeromonas, the chromosomal location of FGIs is based on the detection of three types of genes: genes involved in the bi...

Watch this Scientific Journal Video about Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility at JoVE.com

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...

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Immunology and Infection

2.1K Views.  Universidad de Barcelona. Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

Video: Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility provides X. fastidiosa a means for long-distance intra-plant movement and colonization, contributing toward pathogenicity in X. fastidiosa. The twitching motility of X. fastidiosa is operated by type IV pili. Type IV pili of Xylella fastidiosa are regulated by pilG, a chemotaxis regulator in Pil-Chp operon encoding proteins that are involved with signal transduction pathways. To elucidate the roles of pilG in the twitching motility of X. fastidiosa, a pilG-deficient mutant Xf&#916;pilG and its complementary strain Xf&#916;pilG-C containing native pilG were developed. A microfluidic chambers integrated with a time-lapse image recording system was used to observe twitching motility in Xf&#916;pilG, Xf&#916;pilG-C and its wild type strain. Using this recording system, it permits long-term spatial and temporal observations of aggregation, migration of individual cells and populations of bacteria via twitching motility. X. fastidiosa wild type and complementary Xf&#916;pilG-C strain showed typical twitching motility characteristics directly observed in the microfluidic flow chambers, whereas mutant Xf&#916;pliG exhibited the twitching deficient phenotype. This study demonstrates that pilG contributes to the twitching motility of X. fastidiosa. The microfluidic flow chamber is used as a means for observing twitching motility.

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce&#39;s disease in grapevine.

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...