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><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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent</td><td>10017608</td><td>for broth preparation</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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>&amp;lambda;-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>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent</td><td>10002917</td><td>for broth preparation</td></tr><tr><td>PrimeScript&lt;sup&gt;R&lt;/sup&gt;RT reagent Kit with gDNA Eraser&amp;nbsp;</td><td>TaKaRa</td><td>RR047</td><td>qRT-PCR</td></tr><tr><td>SYBR&lt;sup&gt;R&lt;/sup&gt; 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&amp;nbsp;</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>&amp;nbsp;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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Universidad de Cartagena UDEC

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

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Genetics

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

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 ...

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5&#8217; extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive bacteria.&#160;

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

Biochemistry

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free in the cytosol (cytosolic hyper-replication). These different phenotypes of intracellular replication drive to different pathways of pathogenesis, i.e., cytosolic hyper-replication induces inflammatory cell death and extrusion into the gut lumen, while vacuolar replication leads to trans-epithelium penetration and systemic spread. Significant effort was made to create microscopy tools to study the behavior of Salmonella inside invaded cells, such as the pCHAR-Duo fluorescence reporter plasmid that allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP. However, intracellular phenotypes are often manually scored, a time-consuming procedure that limits analysis to a small number of samples and cells. To overcome these limitations, two complementary and automated image analyses were developed using ImageJ, a freely available image analysis software. In the high-throughput protocol, epithelial cells were infected with Salmonella carrying pCHAR-Duo using 96-well plates. Imaging was performed using an automated fluorescence microscope. Then, two image analysis methods were applied to measure the intracellular behavior of Salmonella at different detail levels. The first method measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication. It is fast and allows the scoring of a high number of cells and samples, making it suitable for high-throughput assays such as screening experiments. The second method performs single-cell analysis to determine the percentage of infected cells, the mean vacuolar load of Salmonella, and the cytosolic hyper-replication rate giving greater details about Salmonella behavior inside epithelial cells. The protocols can be performed by specifically designed ImageJ scripts to automatically run batch analyses of the major steps of Salmonella-enterocyte interaction.

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and scoring.

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free ...

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

Summary

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Chapters in this video

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

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

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ