This study was supported by grants from the Chinese National Science Foundation (Nos. 31972651 and 31101826), Jiangsu High Education Science Foundation (No.14KJB230002), State Key Laboratory of Veterinary Biotechnology (No.SKLVBF201509), Nature Science Foundation Grant of Yangzhou (No.YZ2014019), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

All the experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council. The animal care and use committee of Yangzhou University approved all experiments and procedures applied on the animals (SYXK2016-0020).
1. Bacterial strains, plasmids, and culture conditions
<ol>
	<li>Use the bacteria and plasmids listed in Table 1.</li>
	<li>Culture bacteria in LB broth or on LB agar plates at 37 &#176;C, in the presence of 50 &#181;g/mL ampicillin (Amp) when appropriate.</li>
	<li>Culture strains containing temperature sensitive plasmids are used for deletion mutant construction at 30 &#176;C.</li>
</ol>
2. Clone micC gene of S. Enteritidis strain 50336
<ol>
	<li>Based on the upstream and downstream sequence of micC gene of S. Typhimurium strain SL1344, design primers vmicC-F and vmicC-R to amplify a fragment containing micC gene by PCR using SE50336 genomic DNA as template.</li>
	<li>Mix 5 &#181;L of 10x PCR reaction buffer, 2 &#181;L of dNTP mixture (2.5 mM), 1 &#181;L of vmicC-F and vmicC-R primers, respectively, 5 &#181;L of template, 1 &#181;L of Taq DNA polymerase and 35 &#181;L of ddH2O together for PCR.</li>
	<li>Use the following PCR reaction conditions:pre-denaturation at 94 &#730;C for 4 min; 94 &#730;C for 30 s, 53 &#730;C for 1 min, 72 &#730;C for 1 min for 25 cycles, and extension at 72 &#730;C for 10 min.</li>
	<li>Sequence the PCR product to obtain the micC gene sequence.</li>
</ol>
3. Construction of the micC deletion mutant
NOTE: The micC-negative mutant of Salmonella Enteritidis strain 50336 was constructed using &#955;-Red-mediated recombination as described previously13,14. The primers used are listed in Table 2.
<ol>
	<li>Amplify chloramphenicol cassette containing homology fragments of micC gene.
	<ol>
		<li>Design micC-F and micC-R primers to amplify the chloramphenicol (Cm) cassette from plasmid pKD3, including 50 bp homology extensions from the 5&#39; and 3&#39; of the micC gene.</li>
		<li>Extract the pKD3 plasmid as the PCR template.</li>
		<li>Mix 5 &#181;L of 10x PCR reaction buffer, 2 &#181;L of dNTP Mixture (2.5 mM), 1 &#181;L of micC-F and micC-R primers, respectively, 5 &#181;L of template, 1 &#181;L of Taq DNA polymerase and 35 &#181;L of ddH2O together as PCR reaction mixture</li>
		<li>Amplify the Cm cassette with the following PCR reaction conditions: pre-denaturation at 94 &#176;C for 4 min; 94 &#176;C for 1 min, 52 &#176;C for 1 min, 72 &#176;C for 1 min for 10 cycles; 94 &#176;C for 1 min, 63 &#176;C for 1 min, 72 &#176;C for 1 min for 25 cycles, and extension at 72 &#176;C for 10 min.</li>
		<li>Detect the size of PCR product by agarose gel electrophoresis. Purify and recover PCR product with DNA gel recovery kit, and determine the concentration of DNA by spectrophotometer. 
		CAUTION: PCR must be carried out twice. The first PCR product was diluted at a ratio of 1:200 and used as a template for the secondary PCR, to eliminate the interference of further recombination by pKD3 plasmid.</li>
	</ol>
	</li>
	<li>Construct 1st recombinant strain 50336&#916;micC::cat
	<ol>
		<li>Mix 100 &#181;L of SE50336 competent cells with 5 &#181;L of pKD46 plasmid uniformly and incubate on ice for 30 min. Heat shock the above mixture at 42 &#176;C for 90 s, and rapidly transfer the mixture to ice for 2 min to transform the pKD46 plasmid to SE50336. Screen positive colonies by culturing overnight at 30 &#176;C on an Amp (50 &#181;g/mL) resistant plate.</li>
		<li>Add 30 mM L-arabinose to SE50336/pKD46 liquid culture, and induce recombinase expression by a 30 &#176;C shaking culture for 1 h. Then prepare competent cells.</li>
		<li>Mix 100 ng of purified PCR product (step 3.1) and 40 &#181;L of SE50336/pKD46 competent cells into an electric shock cup (e.g., Bio-Rad). Carry out electric shock transformation with the parameters of voltage 1.8 kV, pulse 25 &#181;F and resistance 200 &#937;.</li>
		<li>After electrotransformation, transfer the mixture to 1 mL of SOC medium and a shaking culture at 150 rpm and 30 &#176;C for 1 h. Then smear the mixture on a Cm (34 &#181;g/mL) resistant LB plate and culture at 37 &#176;C overnight to screen positive colony.</li>
		<li>Culture the above positive colony at 42 &#176;C for 2 h. Screen the colony that is sensitive to Amp (50 &#181;g/mL) but resistant to Cm (34 &#181;g/mL) at 37 &#730;C overnight to obtain the 1st recombinant strain without pKD46.</li>
	</ol>
	</li>
	<li>Identify the 1st recombinant strain 50336&#916;micC::Cat.
	<ol>
		<li>Extract 50336&#916;micC::Cat genomic DNA as the PCR template. Use the same PCR reaction components as in step 2.1. Carry out the PCR reaction with the same conditions as in step 2.1.</li>
		<li>Detect the size of PCR product by agarose gel electrophoresis and sequence the PCR product.</li>
	</ol>
	</li>
	<li>Construct deletion mutant 50336&#916;micC.
	<ol>
		<li>Electroporate 100 ng of plasmid pCP20 into 40 &#181;L of 50336&#916;micC::Cat competent cells with the parameters of voltage 1.8 kV, pulse 25 &#181;F and resistance 200 &#937;, screen positive transformants on both Amp (50 &#181;g/mL) and Cm (34 &#181;g/mL) resistant plate at 30 &#176;C.</li>
		<li>Transfer above positive transformants into non-resistant LB and culture them overnight at 42 &#176;C, and then isolate single colonies on an LB plate at 37 &#176;C. Select the colony that is sensitive to both Amp and Cm. This mutant is the micC deletion mutant SE50336&#916;micC.</li>
		<li>Verify 50336&#916;micC by PCR.
		<ol>
			<li>Extract 50336&#916;micC genomic DNA as PCR template. Mix 5 &#181;Lof 10x PCR reaction buffer, 2 &#181;L of dNTP Mixture (2.5 mM), 1 &#181;L of primer vmicC-F, 1 &#181;L of primer vmicC-R, 5 &#181;L of template, 1 &#181;L of Taq DNA polymerase and 35 &#181;L of ddH2O together for PCR.</li>
			<li>Use the following PCR reaction conditions:pre-denaturation at 94 &#176;C for 4 min; 94 &#176;C for 30 s, 53 &#176;C for 1 min, 72 &#730;C for 1 min for 25 cycles, and extension at 72 &#176;C for 10min.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Construction of the micC complemented strain
<ol>
	<li>Design primers pBR-micC-F and pBR-micC-R with NheI and SalI restriction sites.
	<ol>
		<li>Amplify full-length micC gene with flank sequences using PCR reaction mixture that contains 5 &#181;L of SE50336 genomic DNA as template, primers 1 &#181;L of pBR-micC-F and 1 &#181;L of pBR-micC-R as primers, 5 &#181;Lof 10x PCR reaction buffer, 2 &#181;L of dNTP Mixture (2.5 mM), 2 &#181;L of dNTP Mixture (2.5 mM) and 35 &#181;L of ddH2O.</li>
		<li>Use the following PCR reaction conditions: pre-denaturation at 94 &#176;C for 4 min; 94 &#176;C for 30 s, 52 &#176;C for 50 s, 72 &#176;C for 1 min for 25 cycles, and extension at 72 &#176;C for 10 min. Purify and recover PCR product.</li>
	</ol>
	</li>
	<li>Digest PCR product and plasmid pBR322 respectively using restriction enzyme NheI and SalI, and ligate them using T4 ligase at 16 &#176;C overnight to obtain the plasmid pBR322-micC.</li>
	<li>Transform pBR322-micC into the SE50336&#916;micC competent cells, and screen positive transformant to obtain the complemented strain SE50336&#916;micC/pmicC. Extract plasmid pBR322-micC from complemented strain and verify it by restriction enzyme digestion and sequencing.</li>
</ol>
5. RNA isolation and quantitative real-time PCR
<ol>
	<li>Culture SE50336, 50336&#916;micC, and 50336&#916;micC/pmicC in LB medium overnight at 24 &#176;C with 180 rpm shake cultivation to an OD600 of 2.0. Collect bacterial culture by centrifugation at 13000 rpm for 2 min.</li>
	<li>Extract total RNA using TRIzol reagent. Incubate 50 &#181;L of isolated RNA with 2 &#181;L of DNaseI and 6 &#181;L of 10x buffer at 37 &#176;C for 30 min to remove DNA. Determine RNA quantity by pipetting 1 &#181;L of RNA sample to a micro-spectrophotometer.</li>
	<li>Synthesis of cDNA
	<ol>
		<li>Use 1 &#181;g of total RNA for cDNA synthesis in 20 &#181;L of reverse transcription reaction system (4 &#181;L of 5x buffer, 1 &#181;L of RT Enzyme mix, 1 &#181;L of RT primer mix, 10 &#181;L of total RNA, and 4 &#181;L of ddH2O). Incubate above reaction system at 37 &#176;C for 15 min and then at 85 &#176;C for 5 s.</li>
	</ol>
	</li>
	<li>Design primers based on the sequence of target genes ompA, ompC and ompD. Perform reverse transcription-PCR using a RT reagent kit. The PCR reaction components contain 2.5 &#181;Lof 10x PCR reaction buffer, 1 &#181;L of dNTP mixture (2.5 mM), 1 &#181;L of target gene (ompA, ompC or ompD) primers, 2.5 &#181;L of template, 0.5 &#181;L of Taq DNA polymerase and 17.5 &#181;L of ddH2O.
	<ol>
		<li>Use the following PCR reaction conditions:pre-denaturation at 94 &#176;C for 4 min; 94 &#176;C for 30 s, 60 &#176;C for 1 min, 72 &#176;C for 1 min for 25 cycles, and extension at 72 &#176;C for 10min.</li>
	</ol>
	</li>
	<li>Carry out real-time PCR using SYBR green RT-PCR kit in a RT-PCT instrument in triplicates.
	<ol>
		<li>Use the following PCR reaction components: 10 &#181;L of 2x SYBR buffer, 0.4 &#181;Lof forward primer and reverse primer respectively, 0.4 &#181;L of RoxDye II, 2 &#181;Lof cDNA and 6.8 &#181;L of RNase free H2O.</li>
		<li>Use the following PCR reaction conditions:pre-denaturation at 95 &#176;C for 1 min for one cycle; 95 &#176;C for 5 s, 60 &#176;C for 34 s for 40 cycles.</li>
		<li>Normalize all data to the endogenous reference gene gyrA. Use 2-&#916;&#916;CT method for data quantification15.</li>
	</ol>
	</li>
</ol>
6. Virulence assays
<ol>
	<li>Culture SE50336, 50336&#916;micC and 50336&#916;micC/pmicC in LB medium to early stationary phase (OD600 of 2-3) at 24 &#730;C, harvest by centrifugation, and dilute to appropriate CFU mL-1 in sterile PBS.</li>
	<li>For mice infections, dilute the cultured strains to 10 CFU/200 &#181;L, 102 CFU/200 &#181;L and 103 CFU/200 &#181;L gradient resuspensions. Infect groups of five 6-8 week old Balb/c mice per strain by subcutaneous injection. Inject the control group with 200 &#181;L of physiological saline.</li>
	<li>For chicken infections, dilute above three strains to 107 CFU/200 &#181;L, 108 CFU/200 &#181;L and 109 CFU/200 &#181;L gradient resuspensions. Infect groups of twenty 1-day-old chickens per strain by subcutaneous injection.</li>
	<li>Monitor signs of illness and deaths of experimental animals daily. Calculate the LD50 (median lethal dose) 14 d post-infection as described previously16. Process the data using data analysis software.</li>
	<li>In infection groups, collect the heart, liver, spleen, lung, and kidney of freshly dead chicks. Weigh 0.5 g of the above tissues separately and grind them with sterile operation. Dilute grinding samples gradually, spread them on LB plate and culture for 8-10 h at 37 &#730;C. Record the amount of Salmonella strains colonized in chick tissues.</li>
</ol>

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K., Gottesman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+RyhB+small+RNA+on+global+iron+use+in+Escherichia+coli.">Effect of RyhB small RNA on global iron use in Escherichia coli.</a> Journal of Bacteriology. 187 (20), 6962-6971 (2005).</li><li>Papenfort, K., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+RNA+in+bacterial+pathogens.">Regulatory RNA in bacterial pathogens.</a> Cell Host &amp; Microbe. 8 (1), 116-127 (2010).</li></ol>

The authors have nothing to disclose.

S. Enteritidis is an important facultative intracellular pathogen that can infect young chickens and produces symptoms from enteritis to systemic infection and death17,18. In addition, S. Enteritidis causes latent infections in adult chickens and chronic carriers contaminate poultry products, resulting in food-borne infections in humans19. The pathogenic mechanism of S. Enteritidis remains to be further probed. T...

Non-coding small RNAs (sRNAs) are 40-400 nucleotides in length, which generally do not encode proteins but could be transcribed independently in bacterial chromosomes1,2,3. Most sRNAs are encoded in the intergenic regions (IGRs) between gene-coding regions and interact with target mRNAs through base-pairing actions, and regulate target genes expression at the post-transcriptional level4,5. They play important regulation roles in substance metabolism, outer membrane protein synthesis, quorum sensing and virulence gene expression5.
MicC is a 109-nucleotide small RNA transcript present in Escherichia coli and Salmonella enterica serovar Typhimurium, which could regulate multiple outer membrane protein expression such as OmpC, OmpD, OmpN, Omp35 and Omp366,7,8,9. MicC regulates the expression of OmpC by inhibiting ribosome binding to the ompC mRNA leader in vitro and it requires the Hfq RNA chaperone for its function in Escherichia coli6. In Salmonella Typhimurium, MicC silences ompD mRNA via a &#8804;12-bp RNA duplex within the coding sequence (codons 23-26) and then destabilizes endonucleolytic mRNA7. This regulation process is assisted by chaperone protein Hfq10. The OmpC is an abundant outer membrane protein that was thought to be important in environments where nutrient and toxin concentrations were&#160;high, such as in the intestine6. The OmpD porin is the most abundant outer membrane protein in Salmonella Typhimurium and represents about 1% of total cell protein11. OmpD is involved in adherence to human macrophages and intestinal epithelial cells12. MicC also represses the expression of both OmpC and OmpD porins. It is thought that MicC may regulate virulence. To explore new target genes regulated by MicC and study the virulence regulation function of micC, we cloned the micC gene in the Salmonella Enteritidis (SE) strain 50336, and then constructed the mutant 50336&#916;micC and the complemented mutant 50336&#916;micC/pmicC. Novel target genes were screened by qRT-PCR. The virulence of 50336&#916;micC was detected by mice and chicken infections.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>dextrose</td>
			<td>Sangon Biotech</td>
			<td>A610219</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>DNA purification kit</td>
			<td>TIANGEN</td>
			<td>DP214</td>
			<td>for DNA purification</td>
		</tr>
		<tr>
			<td>Ex Taq</td>
			<td>TaKaRa</td>
			<td>RR01A</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10017608</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>20032116</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>L-Arabinose</td>
			<td>Sangon Biotech</td>
			<td>A610071</td>
			<td>&#955;-Red recombination</td>
		</tr>
		<tr>
			<td>Mini Plasmid Kit</td>
			<td>TIANGEN</td>
			<td>DP106</td>
			<td>plasmid extraction</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019308</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10002917</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>PrimeScriptRRT reagent Kit with gDNA Eraser&#160;</td>
			<td>TaKaRa</td>
			<td>RR047</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>SYBRR Premix Ex Taq II</td>
			<td>TaKaRa</td>
			<td>RR820</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202</td>
			<td>Ligation</td>
		</tr>
		<tr>
			<td>TRIzol&#160;</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf</td>
			<td>5418</td>
			<td>centrifugation</td>
		</tr>
		<tr>
			<td>Electrophoresis apparatus</td>
			<td>Bio-Rad</td>
			<td>164-5050</td>
			<td>Electrophoresis</td>
		</tr>
		<tr>
			<td>&#160;Electroporation System</td>
			<td>Bio-Rad</td>
			<td>165-2100</td>
			<td>for bacterial transformation</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>BioTek</td>
			<td>Epoch</td>
			<td>Absorbance detection</td>
		</tr>
		<tr>
			<td>Real-Time PCR system</td>
			<td>Applied Biosystems</td>
			<td>7500 system</td>
			<td>qRT-PCR</td>
		</tr>
	</tbody>
</table>

a non-coding small rna micc contributes to virulence in outer membrane proteins in salmonella enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336&#916;micC by the &#955; Red-based recombination system and the complemented mutant 50336&#916;micC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336&#916;micC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336&#916;micC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336&#916;micC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336&#916;micC and 50336&#916;micC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

Construction of the mutant 50336&#916;micC and complemented strain 50336&#916;micC /pmicC 
The micC gene clone result indicated that this gene was composed of 109 bp showing 100% identity with that of S. Typhimurium. Based on the sequence data, the deletion mutant 50336&#916;micC and the complemented mutant 50336&#916;micC/pmicC w...

Watch this Scientific Journal Video about A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis at JoVE.com

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An &#955;-Red-mediated recombination system was used to create a deletion mutant of a small non-coding RNA micC.

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia ...

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Research

JoVE Journal

Immunology and Infection

1.6K Views.  Yangzhou University. An λ-Red-mediated recombination system was used to create a deletion mutant of a small non-coding RNA micC. 

Video: A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...