This work was supported by National Health and Medical Research Council (NHMRC) grants (Grant ID 1062383 and 1063646) awarded to HJN. EAL is supported by an Australian Postgraduate Award.

The authors have nothing to disclose.

Secretion systems, and the bacterial effector proteins these systems transport into the cytoplasm of host cells, are an important virulence component that many pathogenic bacteria utilize to establish an infection within unique replicative niches. The focus of many research groups has been to investigate the interplay between bacterial effectors and host proteins and the influence these effectors have on host cellular pathways. Very limited research, if any, has examined the potential for host proteins to be necessary for successful bacterial effector translocation.
In this study we used the obligate, intracellular pathogen Coxiella burnetii, the causative agent of zoonotic Q fever. After entry into host cells, the Coxiella-containing vacuole passively traffics through the canonical endocytic pathway until reaching an acidic lysosome-derived vacuole, where it replicates to very high numbers. Apart from taking advantage of the normal host trafficking pathway, the ability of Coxiella to establish a successful replicative niche also relies on a functional type IVB secretion system (T4SS) and subsequent effector translocation8,17. This secretion system is functionally analogous to the well-characterized Dot/Icm T4SS of Legionella. This was aptly demonstrated by complementing specific dot/icm mutants of Legionella with their respective Coxiella genes19,20. Although the T4SSs of both Legionella and Coxiella are essential for replication, the timing of when these systems are activated is quite different and reflects the divergent nature of their respective replicative niches. The Legionella T4SS is triggered immediately upon contact with host cells and the rapid translocation of effectors allows the bacteria to avoid the default endosomal-lysosomal pathway and instead create a unique ER-derived replicative vacuole42. In contrast, the T4SS of Coxiella is not activated until approximately 8 hr post-infection, after the bacteria reach the acidic lysosome-derived vacuole21.
Given that Coxiella passively transits through the endocytic pathway and the translocation system is not activated until it reaches the lysosome, a unique opportunity presents itself to use Coxiella as a tool to study endocytic maturation. The requirement for the CCV to become acidified before effectors are translocated indicates that a number of host proteins are important for the translocation of effector proteins into the host cytoplasm, in particular, but not limited to, proteins involved in the endocytic trafficking pathway. Utilizing siRNA in a specific targeted manner we can begin to elucidate the role of particular host proteins on effector translocation. In this study we focused on two proteins that regulate the eukaryotic endocytic trafficking pathway, and hence were predicted to effect translocation of the Coxiella effector CBU0077. Rab5A and Rab7A control membrane fusion with early and late endosomes, respectively. Silencing either of these proteins using siRNA resulted in a significant decrease in translocation efficiency of CBU0077 when compared to the control OTP siRNA (Figure 5B). The representative results shown here reflect our previous published findings21 and provide further validation to the importance of these human Rab proteins to Coxiella effector translocation. This technique has also been employed to characterize the impact of other host processes on Coxiella Dot/Icm effector translocation. The retromer complex was found to be important for intracellular replication of Coxiella. By silencing retromer components, using siRNA, and then conducting a translocation assay we could show that retromer is required to create the environment that induces Coxiella effector translocation 43.
Although the representative results shown here is a comparison between infected OTP control and siRNA targeting Rab5A and Rab7A, the protocol can be modified according to experimental needs. For example, the level of effector translocation can also be compared to cells transfected with scrambled siRNA or non-transfected cells.
The focus of this particular study was the obligate, intracellular pathogen C. burnetii and proteins involved in endocytic trafficking, however, the methods described within can be easily adapted to other intracellular and extracellular pathogens that utilize secretion systems for virulence. Indeed, the fluorescent-based translocation assay that relies on &#946;-lactamase activity within the host cytoplasm used here, has already been extensively used across a range of pathogens30-32,35,44. Naturally, the infection conditions of the specific cell lines used must be adapted to reflect the infection requirements of specific bacteria. Additionally, a known endogenous effector fused to &#946;-lactamase is paramount to the success of the methods described within. One important consideration for successful implementation of the protocol explained here is the impact silencing a particular host target could have on bacterial entry and subsequent replication. Here, effector translocation by Coxiella is measured 24 hr post infection. In this instance, and for other intracellular bacteria, the ability of the organism to gain entry into host cells is vital. Additionally, specific gene silencing could also influence bacterial replication therefore altering the level of translocation observed. Confirmation that siRNA silencing does not significantly impact bacterial entry and/or replication, and consequently translocation efficiency, could be achieved using either microscopy or numeration of bacteria. The translocation data can then be normalized for the number of infected cells per siRNA treatment. Bacterial replication has not confounded the data presented here as Coxiella undergoes little to no replication during the 24 hr incubation period.
This method requires efficient transfection of siRNA into host cells to ensure sufficient silencing of the gene of interest. With this in mind, choosing the correct transfection reagent is extremely important. Apart from the reagent and conditions used in this particular study, which has been optimized for HeLa 229 cells, there are numerous other reagents to select from. The knockdown efficiency using different transfection reagents can be quantified using RT-qPCR to ensure that both the most appropriate reagent is selected and that the siRNA chosen specifically targets the transcript of interest. Although not presented in this study, we have previously demonstrated the knockdown efficiency of both Rab5A and Rab7A using RT-qPCR21. Additionally, the use of antibodies against the target of interest and confirmation by western blot can also confirm siRNA silencing of specific targets.
Two other important considerations to take note of when using siRNA include the possibility of off-target effects and the cell viability of transfected cells. Off-target effects occur when unintended targets are also destroyed. This can lead to phenotypic changes and might result in false positives. In this study we utilized a pool of four siRNA duplexes to silence a single host target. If silencing a particular target causes a reduction in translocation efficiency, validation that this result is not due to off-target effects can be achieved by separating the four siRNA duplexes and repeating the translocation assay. At least two out of four siRNA duplexes should demonstrate a similar phenotype to the original siRNA pool. With this result, one can be highly confident that the observed translocation efficiency is not due to off-target effects. The validity of the translocation results produced is also dependent on cell viability. The application of a nuclei stain after the translocation assay enables enumeration of cell viability following siRNA silencing. In the case of PLK1, significant cell death can be easily observed without staining; however, using epifluorescence microscopy a more precise representation can be achieved. If by silencing a particular host target significant cell death is observed, for example 50 % compared to the control, it is unlikely that an accurate picture can be inferred as to the importance of that particular host protein to the translocation of effectors. As shown in this study, although silencing either Rab5A or Rab7A has a slight impact on cell viability (Figure 5C), this reduction is not pronounced enough to negate the validity of the translocation data observed. Another consideration with regards to cell viability following siRNA silencing is the observation that significant cell death often leads to an increase in the translocation ratio. This is aptly demonstrated by the translocation data reported for PLK1 (Figure 5A). The reduced cell number results in a proportional increase in the multiplicity of infection. This will increase the infection rate and hence the translocation ratio.
Although not presented in this study, visualization of effector translocation can also yield a complimentary quantitative result. Using an epifluorescent microscope fitted with a long-pass dichroic mirror to separate excitation and emission light can provide a visual observation of intracellular BlaM substrate fluorescence. A possible alternative to the &#946;-lactamase reporter system is to utilize the adenylate-cyclase reporter system. Similar to the reporter system described here, the gene of interest is fused to a calmodulin-dependent adenylate cyclase gene (cyaA) derived from Bordetella pertussis. Effector translocation can be measured by quantification of intracellular cyclic AMP (cAMP) using an immunoassay ELISA kit. Since the activity of CyaA is entirely dependent on host cell calmodulin, which is only present in the host cytosol, effector translocation is confirmed by an increase in cAMP levels. Although this method has also been used successfully to measure effector translocation for a variety of bacteria45-47, quantification of intracellular cAMP relies on the lysis of host cells to extract cAMP, a process that is more timeconsuming compared to the method described within. Measurement of effector translocation using the FRET-based &#946;-lactamase reporter system is more efficient since the BlaM substrate can be added directly to the cells and fluorescence can be measured within hours. Additionally, the method described here can be more easily adapted to generate high-throughput results, especially in a 96-well format.
Many bacterial pathogens depend on protein translocation systems to introduce effector proteins into host cells allowing modulation of host cell functions to benefit the pathogen. Type IV secretion systems are used by intracellular bacteria such as Legionella, Coxiella and Brucella and Type III secretion systems are utilized by a range of pathogens including Chlamydia, attaching and effacing E. coli and Salmonella. By comparing the effect of silencing expression of specific host proteins on effector translocation by a range of pathogens, an understanding of host factors necessary for effector translocation by particular types of secretion systems or specific to an individual pathogen could be elucidated. This provides a unique approach to study the intricacies of host-pathogen interactions.

Coxiella burnetii is a unique intracellular pathogen that causes the zoonotic human infection Q fever. This disease is associated with a broad spectrum of clinical presentations extending from asymptomatic seroconversion to life-threatening chronic infection that often manifests as endocarditis years after exposure1. Human infection occurs primarily through the inhalation of contaminated aerosols with ruminants the major reservoir for infection, in particular, dairy cows, sheep and goats. Although Coxiella infection in these animals is typically subclinical, the infection may trigger abortion and the considerable bacterial load within the birthing fluid and placenta can contaminate the local environment1. An example of the enormous burden such contamination can have on both public health and the agriculture industry was recently observed in the Q fever outbreak that occurred in the Netherlands2. Between 2007 and 2010, over 4,000 human cases of Q fever were diagnosed and this outbreak was linked to significant contamination of goat farms3. Additionally, Coxiella is a potential biological weapon, as classified by the US Centers for Disease Control and Prevention, due to the environmental stability of the bacteria and low infectious dose necessary to cause severe morbidity and mortality4.
Coxiella exists in two phases: Phase I organisms, isolated from natural sources, are extremely virulent and Phase II organisms are highly attenuated in vivo. For example, after several in vitro passages of Coxiella burnetii Nine Mile Phase I organisms, Phase II bacteria were produced that contain an irreversible chromosomal deletion resulting in truncated lipopolysaccharide (LPS)5. This strain, C. burnetii NMII, is phenotypically similar to Phase I in tissue culture models and provides a safer model for researchers to study Coxiella pathogenesis in laboratories5. In recent years several breakthroughs have rapidly advanced the field of Coxiella genetics. Most notably, the development of axenic media (acidified citrate cysteine medium - ACCM-2) has allowed the cell-free growth of Coxiella in both liquid and on solid media6,7. This resulted in direct improvements of genetic tools available for Coxiella including an inducible gene expression system, shuttle vectors and random transposon systems8-11. Most recently, two methods for targeted gene inactivation have also been developed, paving the way for examining specific virulence gene candidates12.
Following internalization by alveolar macrophages, Coxiella replicates to high numbers within a membrane-bound compartment termed the Coxiella-containing vacuole (CCV). The CCV requires host endocytic trafficking through early and late endosomes until it matures into a lysosome-derived organelle13. Throughout this process, the CCV acquires host factors that either appear transiently or remain associated with the vacuole, including, but not limited to, Rab5, Rab7, CD63 and LAMP-113-15. Replication of Coxiella within host cells is entirely dependent on a fully functional Dot/Icm Type IVB Secretion System (T4SS)8,16,17. This secretion system is a multi-protein structure ancestrally related to conjugation systems and spans both bacterial and vacuolar membranes to deliver bacterial proteins, termed effectors, into the host cytoplasm18. The Coxiella T4SS is functionally very similar to the well characterized Type IVB Dot/Icm Secretion System of Legionella pneumophila19,20. Interestingly, activation of the T4SS and subsequent effector translocation does not occur until Coxiella reaches the acidic lysosome-derived organelle, approximately 8 hr post-infection17,21. To date, over 130 Dot/Icm effectors have been identified9,17,22-24. Many of these effectors likely play important roles during replication of Coxiella within host cells; however, only a few effectors have been functionally characterized25-29.
In this study we utilize a fluorescence based translocation assay that relies on cleavage of the CCF2-AM FRET substrate (hereafter referred to as the BlaM substrate) via &#946;-lactamase activity within the host cell cytoplasm (Figure 1). The gene of interest is fused to TEM-1 &#946;-lactamase (BlaM) on a reporter plasmid that provides constitutive expression. The BlaM substrate is composed of two fluorophores (coumarin and fluorescein) that form a FRET pair. Excitation of the coumarin results in FRET of the fluorescein and green fluorescence emission in the absence of effector translocation; however, if the BlaM-effector fusion protein is translocated into the host cytoplasm, the resultant &#946;-lactamase activity cleaves the &#946;-lactam ring of the BlaM substrate, separating the FRET pair producing blue fluorescence emission following excitation. This translocation assay has been well proven as an approach to identify effector proteins from a range of different intracellular and extracellular bacteria, including C. burnetii, L. pneumophila, L. longbeachae, Chlamydia trachomatis, enteropathogenic E. coli, Salmonella and Brucella17,30-35.
To determine the role of specific host factors on Coxiella effector translocation we utilize a well-established method for gene silencing known as RNA interference, in particular small interfering RNA (siRNA). Originally identified in Caenorhabditis elegans, RNA interference is a conserved endogenous cellular process used for innate defense against viruses as well as gene regulation36,37. After binding of sequence-specific siRNA, degradation of mRNA occurs through RISC (RNA-induced silencing complex) resulting in specific gene silencing or knockdown38. In this study, siRNA was used to target two host proteins, Rab5A and Rab7A, which are important regulators of the endocytic pathway. The impact of silencing Rab5A and Rab7A on effector translocation was ascertained using C. burnetii pBlaM-CBU0077. CBU0077 was selected as it was previously shown to be translocated by the Dot/Icm secretion system of Coxiella17.
Utilizing both siRNA gene silencing and the fluorescencebased translocation assay described here, we are beginning to establish a role for host factors in the translocation of effector proteins by Coxiella. This approach can be applied to a wide range of both intracellular and extracellular bacteria that possess similar secretion systems responsible for the translocation of effector proteins.

<table border="0" cellpadding="0" cellspacing="0" width="760">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Reagents</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Citric acid</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">C0759</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium citrate</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">S4641</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Potassium phosphate</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">60218</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Magnesium chloride</td>
			<td style="width:87px;">Calbiochem</td>
			<td style="width:119px;">442611</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Calcium chloride</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">C5080</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Iron sulfate</td>
			<td style="width:87px;">Fisher</td>
			<td style="width:119px;">S93248</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium chloride</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">S9625</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">L-cysteine</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">C6852</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Bacto-neopeptone</td>
			<td style="width:87px;">BD</td>
			<td style="width:119px;">211681</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Casamino acids</td>
			<td style="width:87px;">Fisher</td>
			<td style="width:119px;">BP1424</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Methyl beta cyclodextrin</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">C4555</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">RPMI + Glutamax</td>
			<td style="width:87px;">ThermoFisher Scientific</td>
			<td style="width:119px;">61870-036</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Chloramphenicol</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">C0378</td>
			<td style="width:211px;">For bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">ON-TARGETplus Non-targeting Control Pool (OTP)</td>
			<td style="width:87px;">Dharmacon</td>
			<td style="width:119px;">D-001810-10-05</td>
			<td style="width:211px;">Non-targeting control</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">siGENOME Human PLK1 (5347) siRNA - SMARTpool</td>
			<td style="width:87px;">Dharmacon</td>
			<td style="width:119px;">M-003290-01-005</td>
			<td style="width:211px;">Causes cell death; measure of transfection efficiency</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">siGENOME Human RAB5A (5968) siRNA - SMARTpool</td>
			<td style="width:87px;">Dharmacon</td>
			<td style="width:119px;">M-004009-00-0005</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">siGENOME Human RAB7A (7879) siRNA - SMARTpool</td>
			<td style="width:87px;">Dharmacon</td>
			<td style="width:119px;">M-010388-00-0005</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:343px;">5X siRNA buffer</td>
			<td style="width:87px;">Dharmacon</td>
			<td style="width:119px;">B-002000-UB-100</td>
			<td style="width:211px;">Use sterile RNase-free water to dilute 5X siRNA buffer to 1X siRNA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DharmaFECT-1 Transfection Reagent</td>
			<td style="width:87px;">Dharmacon</td>
			<td style="width:119px;">T-2001-01</td>
			<td style="width:211px;">For transfection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Opti-MEM + GlutaMAX</td>
			<td style="width:87px;">ThermoFisher Scientific</td>
			<td style="width:119px;">51985-034</td>
			<td style="width:211px;">Reduced-serum medium used for transfection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">DMEM + GlutaMAX</td>
			<td style="width:87px;">ThermoFisher Scientific</td>
			<td style="width:119px;">10567-014</td>
			<td style="width:211px;">For cell culture and infection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Heat-inactivated fetal calf serum (FCS)</td>
			<td style="width:87px;">Thermo Scientific</td>
			<td style="width:119px;">SH30071.03</td>
			<td style="width:211px;">Can use alternate equivalent product</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DMSO</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td style="width:211px;">For storage of Coxiella strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">PBS</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;">For cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">0.05% Trypsin + EDTA</td>
			<td style="width:87px;">ThermoFisher Scientific</td>
			<td style="width:119px;">25300-054</td>
			<td style="width:211px;">For cell culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:343px;">dH2O</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;">For dilution of samples and standards for qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Quick-gDNA Mini Prep</td>
			<td style="width:87px;">ZYMO Research</td>
			<td style="width:119px;">D3007</td>
			<td style="width:211px;">Can use alternate equivalent product to extract gDNA</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:343px;">SensiFAST SYBR No-ROX Kit</td>
			<td style="width:87px;">Bioline</td>
			<td style="width:119px;">BIO-98020</td>
			<td style="width:211px;">Can use alternate equivalent qPCR master mix product for qPCR reaction</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:343px;">LiveBLAzer FRET-B/G Loading kit with CCF2-AM</td>
			<td style="width:87px;">Invitrogen</td>
			<td style="width:119px;">K1032</td>
			<td style="width:211px;">For measurement of translocation using fluorescence and generation of the 6X loading solution (contains Solution A, B, C and DMSO)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium hydroxide</td>
			<td style="width:87px;">Merck</td>
			<td style="width:119px;">106469</td>
			<td style="width:211px;">Probenicid solution component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium phosphate monobasic</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">71505</td>
			<td style="width:211px;">Probenicid solution component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">di-Sodium hydrogen orthophosphate</td>
			<td style="width:87px;">Merck</td>
			<td style="width:119px;">106586</td>
			<td style="width:211px;">Probenicid solution component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Probenicid</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">P8761</td>
			<td style="width:211px;">Probenicid solution component</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:343px;">DRAQ5 Fluorescent Probe Solution (5 mM)</td>
			<td style="width:87px;">ThermoFisher Scientific</td>
			<td style="width:119px;">62251</td>
			<td style="width:211px;">Nuclei stain to determine cell viablity. Use 1:4000 diluted in PBS.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:343px;">4% PFA (paraformaldehyde) solution in PBS</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">P6148</td>
			<td style="width:211px;">Fixing solution for HeLa 229 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:343px;">25cm2 tissue culture flask with vented cap</td>
			<td style="width:87px;">Corning</td>
			<td style="width:119px;">430639</td>
			<td style="width:211px;">For growth of bacterial strain</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:343px;">96 Well Flat Clear bottom Black Polystyrene TC-Treated Microplates, Individually wrapped, with Lid, Sterile</td>
			<td style="width:87px;">Corning</td>
			<td style="width:119px;">3603</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:343px;">75cm2 tissue culture flask with vented cap</td>
			<td style="width:87px;">Corning</td>
			<td style="width:119px;">430641</td>
			<td style="width:211px;">For growth of HeLa 229 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:343px;">175cm2 tissue culture flask with vented cap</td>
			<td style="width:87px;">Corning</td>
			<td style="width:119px;">431080</td>
			<td style="width:211px;">For growth of HeLa 229 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Haemocytometer</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;">For quantification of HeLa 229 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Tear-A-Way 96 Well PCR plates</td>
			<td style="width:87px;">4titude</td>
			<td style="width:119px;">4ti-0750/TA</td>
			<td style="width:211px;">For qPCR reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">8-Lid chain, flat</td>
			<td style="width:87px;">Sarstedt</td>
			<td style="width:119px;">65.989.002</td>
			<td style="width:211px;">For qPCR reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Equipment</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Bench top microfuge</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Bench top vortex</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Orbital mixer</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Centrifuge</td>
			<td style="width:87px;">Eppendorf</td>
			<td style="width:119px;">5810 R</td>
			<td style="width:211px;">For pelleting bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Nanodrop</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;">For gDNA quantification</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:343px;">Mx3005P QPCR machine</td>
			<td style="width:87px;">Aligent Technologies</td>
			<td style="width:119px;">401456</td>
			<td style="width:211px;">For quantification of Coxiella genomes in 7 day culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">ClarioSTAR microplate reader</td>
			<td style="width:87px;">BMG LabTech</td>
			<td style="width:119px;"></td>
			<td style="width:211px;">For measurement of fluorescence</td>
		</tr>
	</tbody>
</table>

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.

For this study, the C. burnetii pBlaM-CBU0077 strain was selected as CBU0077 has been previously shown to be a translocated effector of the Coxiella Dot/Icm secretion system17. Prior to infection, the total number of genomes/ml in the seven day C. burnetii pBlaM-CBU0077 culture was enumerated using qPCR. Figure 4 demonstrates an example of the cycle threshold (Ct) values expected from both standards and samples following qPCR. The Ct values (represented graphically in the Amplification Plot (Figure 4B) and numerically (Figure 4C)) were exported and analyzed as described in 4.3.3. Based on the representative data shown, there were 2.31 &#215; 108 genomes/ml in the 10 ml re-suspended bacterial culture (Figure 4D). To generate the appropriate bacterial dilution (1.88 &#215; 108 genomes/ml in 2 ml), 1.63 ml of resuspended bacteria was added to 370 &#181;l of fresh, pre-warmed DMEM + 10% FCS. Cells reverse transfected with siRNA were subsequently infected with C. burnetii pBlaM-CBU0077 at an MOI of 300 for 24 hr.
After incubation of the reverse transfected infected cells with BlaM substrate quantification of effector translocation can be determined using a fluorescence plate reader. Following analysis as described in 7, all ratio values that have been normalized to uninfected OTP can also be normalized to infected OTP. The level of effector translocation after silencing of host genes can be grouped into three outcomes after comparison to infected OTP control: (1) No change; (2) Increased effector translocation; (3) Decreased effector translocation. Subsequent statistical analysis, for example a Student ttest, can be used to determine the significance of any observed difference to infected OTP control. The result of silencing either Rab5A or Rab7A on effector translocation from three independent experiments is shown in Figure 5A. A significant decrease in the level of BlaM-CBU0077 translocation occurred during silencing of both Rab5A and Rab7A compared to OTP control (p value = 0.0088 (Rab5A) and p value = 0.0059 (Rab7A), paired, Student ttest). The impact of siRNA treatment on cell viability was also established. Following the translocation assay, cells were fixed, stained with a nuclei stain and subsequently quantified. As shown in Figure 5C, although treatment with either Rab5A or Rab7A results in a reduction in cell viability compared to OTP control (12 % and 31 %, respectively), this reduction is not as severe as PLK1 (97 %), a gene known to cause cell death (p-value = 0.0102 (Rab5A); p-value = 0.0081 (Rab7A); p-value &#60; 0.0001 (PLK1), paired, Student t-test). These results demonstrate how silencing of host genes using siRNA can negatively alter the levels of bacterial effector translocation.
<img alt="Figure 1" src="/files/ftp_upload/54210/54210fig1.jpg" /> 
Figure 1.&#160;The Mechanism of Action of the BlaM Substrate During Infection. C. burnetii is internalized by host cells and forms a lysosome-derived vacuole termed the Coxiella-containing vacuole. 24 hr post-infection, cells are loaded with the membrane permeable BlaM substrate that possesses two fluorophores forming a FRET pair (A). In the absence of &#946;-lactamase i.e., no translocation of the BlaM-CBU0077 effector, excitation of the coumarin at 410 nm results in FRET to fluorescein, observed as a green fluorescence signal (520 nm) (B). In the event of a functional Dot/Icm secretion system, the BlaM-CBU0077 fusion protein will be translocated into the host cell and will cleave the BlaM substrate, via &#946;-lactamase activity, causing the separation of the two dyes and resulting in FRET disruption and a blue fluorescence signal (450 nm) after excitation at 410 nm (C). Both the green and blue fluorescence signals can be detected using a fluorescence plate reader. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54210/54210fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54210/54210fig2.jpg" /> 
Figure 2.&#160;Schematic Overview of the Reverse Transfection and Translocation Assay Workflow. Day 1: Prepare the C. burnetii pBlaM-CBU0077 culture. Day 4: Reverse transfect using 40 nM siRNA and seed HeLa 229 cells at a final density of 3.92 &#215; 103 cells/well into a 96-well tray. Day 5: Change media. Day 7: Quantify the total genomes/ml of the C. burnetii pBlaM-CBU0077 culture using qPCR and subsequently infect reverse transfected cells with an MOI of 300. Day 8: Add 6x loading dye, measure fluorescence and quantify cell number. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54210/54210fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54210/54210fig3.jpg" /> 
Figure 3.&#160;The 96-well Plate Layout Used to Set up the Reverse Transfection and Seeding of HeLa 229 Cells. The &#34;blank&#34; wells (shown in red) contain media only and do not need the addition of either siRNA or HeLa 229 cells. The OTP siRNA (shown in light blue for uninfected wells and dark blue for infected wells) is used as a non-targeting control, since it has been optimized to have fewer off-target effects. The maroon and green wells represent the Rab5A and Rab7A siRNA, respectively. PLK1 siRNA (shown in yellow) is used as a measure of transfection efficiency as loss of PLK1 expression can induce pro-apoptotic pathways and extensive cell death in a vast majority of cell lines. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54210/54210fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54210/54210fig4.jpg" /> 
Figure 4.&#160;Representative qPCR Ct Values Used to Quantitate the Total Number of genomes/ml in C. burnetii pBlaM-CBU0077 Culture. (A) The cycle conditions used in the qPCR to enumerate the number of genomes/ml in Coxiella cultures. The Ct values for both standards and samples are represented in graphical (B) and numerical (C) formats. (D) The standard Ct values were averaged, graphed and a logarithmic trendline was calculated. Using the equation and the averages of the sample Ct values the total number of genomes/ml in the C. burnetii pBlaM-CBU0077 culture was determined to be 2.31 &#215; 108. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54210/54210fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54210/54210fig5.jpg" /> 
Figure 5.&#160;Translocation of the Dot/Icm Effector BlaM-CBU0077 during siRNA Silencing of Rab5A and Rab7A. HeLa 229 cells were reverse transfected with siRNA specific to Rab5A (maroon bars), Rab7A (green bars), OTP (blue bars) or PLK1 (yellow bars) and incubated for 72 hr before infection with the C. burnetii pBlaM-CBU0077 strain at an MOI of 300 for 24 hr. After the addition of the BlaM substrate, fluorescence was measured using a microplate reader. (A) The table demonstrates the raw fluorescence values obtained from a single experiment alongside the 450:520 nm ratio relative to uninfected OTP control after subtraction of the blank values (media only, no cells). The level of translocation (B) and cell viability (C) are presented as the percentage mean &#177; standard deviation relative to OTP reverse transfected infected cells from three independent experiments. * p-value &#60; 0.05; **p-value &#60; 0.01; **** p-value &#60; 0.0001 (paired, Student t-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54210/54210fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>13.4 mM</td>
		</tr>
		<tr>
			<td>sodium citrate</td>
			<td>16.1 mM</td>
		</tr>
		<tr>
			<td>potassium phosphate</td>
			<td>3.67 mM</td>
		</tr>
		<tr>
			<td>magnesium chloride</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>calcium chloride</td>
			<td>0.02 mM</td>
		</tr>
		<tr>
			<td>iron sulfate</td>
			<td>0.01 mM&#160;</td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>125.4 mM</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>1.5 mM</td>
		</tr>
		<tr>
			<td>neopeptone</td>
			<td>0.1 g/L</td>
		</tr>
		<tr>
			<td>casamino acids</td>
			<td>2.5 g/L</td>
		</tr>
		<tr>
			<td>methyl beta cyclodextrin</td>
			<td>1 g/L</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>125 ml/L</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;The Components Required to Make 1x ACCM-2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Target concentration</td>
		</tr>
		<tr>
			<td></td>
			<td>1&#181;M</td>
			<td>5 &#181;M</td>
			<td>10&#181;M</td>
			<td>20&#181;M</td>
		</tr>
		<tr>
			<td>Starting Moles</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 nmol</td>
			<td>1 ml</td>
			<td>200 &#181;l</td>
			<td>100 &#181;l</td>
			<td>50 &#181;l</td>
		</tr>
		<tr>
			<td>2 nmol</td>
			<td>2 ml</td>
			<td>400 &#181;l</td>
			<td>200 &#181;l</td>
			<td>100 &#181;l</td>
		</tr>
		<tr>
			<td>5 nmol</td>
			<td>5 ml</td>
			<td>1 ml</td>
			<td>500 &#181;l</td>
			<td>250 &#181;l</td>
		</tr>
		<tr>
			<td>10 nmol</td>
			<td>10 ml</td>
			<td>2 ml</td>
			<td>1 ml</td>
			<td>500 &#181;l</td>
		</tr>
		<tr>
			<td>20 nmol</td>
			<td>20 ml</td>
			<td>4 ml</td>
			<td>2 ml</td>
			<td>1 ml</td>
		</tr>
	</tbody>
</table>
Table 2.&#160;Volume of 1x siRNA Buffer Required for Hydrating Lyophilized siRNA to the Appropriate Concentration.

Watch this Scientific Journal Video about Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing at JoVE.com

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.

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 ...

applying-fluorescence-resonance-energy-transfer-fret-to-examine

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.6K Views.  University of Melbourne. 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.

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

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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 ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...