This work was supported by grant R01 AI141958 from the United States National Institutes of Health/NIAID. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Scalable In Vitro Cultivation of T. pallidum Using Mammalian Cell Co-Culture

<ol>
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The authors have no conflicts of interest to disclose.

The Sf1Ep-TpCM-2 system is the first available procedure that promotes the continuous in vitro culture of T. pallidum. The system is complex due to the extreme growth requirements of this organism: 1) complex nutritional needs because of limited biosynthetic capabilities; 2) a poorly understood requirement for small quantities of oxygen, despite high sensitivity to reactive oxygen species9,10,16,36; and 3) the current need for the presence of Sf1Ep cells. While it is tempting to &#39;cut corners&#39; on the procedure, it is recommended that the steps be followed carefully until a successful long-term culture is achieved prior to trying modifications. As additional information about the metabolic requirements of T. pallidum accumulates, it may be possible to develop axenic conditions that do not require the presence of Sf1Ep cells. However, the in vitro growth rate will likely remain slow (with a minimum doubling time of 33 h to 46 h, depending on the strain)17,18, given that T. pallidum multiplies at an estimated doubling time of 30 h to 33 h even during mammalian infection37,38. As with any bacterial culture, it is recommended that low passage stocks be maintained and that experiments be carried out with T. pallidum cultures that are less than 10 passages from these stocks to avoid &#39;genetic drift&#39; and associated phenotypic changes due to mutations.
Sf1Ep cells apparently provide essential nutrients or enzymatic activities to the treponemes. However, they also consume nutrients (such as glucose and oxygen) and may produce toxic conditions such as low pH9,16,39. Therefore, there is a balancing act between having sufficient Sf1Ep cells to support T. pallidum multiplication and preventing mammalian cell overgrowth and toxicity. High passage numbers of Sf1Ep cells tend to be faster growing and at times, lose the ability to support T. pallidum multiplication. As such, Sf1Ep passage number should be monitored, and cell stocks should be replaced with low-passage frozen preparations periodically. The presence of Sf1Ep cells also complicates the study of T. pallidum properties such as DNA, RNA, and protein content, and enzyme activities. Removal of the rabbit cells is possible to some extent using repeated low-speed centrifugation (100 x g for 5 min) or more effectively using Percoll or Hypaque gradients40,41. However, the gradient centrifugation methods are generally only effective with high numbers of T. pallidum. Alternative methods for propagating T. pallidum are limited to infection of laboratory animals such as rabbits13,14. This approach has ethical considerations and has become increasingly expensive; however, the rabbit model is very useful for studying T. pallidum pathogenesis and host immune responses. In addition, there are likely some differences in gene expression, growth or behavior of T. pallidum during in vitro culture and rabbit infection27.
At the time of this report, the Sf1Ep-TpCM-2 system has been established in at least 6 research groups in the United States and Europe and has resulted in 16 publications with topics ranging from T. pallidum basic biology and genetics to antimicrobial susceptibility. The value of in vitro culture in studying this enigmatic pathogen will likely increase with expanded usage and future improvements.

Treponema pallidum is a species of spiral-shaped bacteria (called spirochetes) that causes syphilis and related infections in humans and other primates. Syphilis is a serious disease with long-term effects on infected individuals, and it is estimated that over 8 million new cases of syphilis occur worldwide each year1. T. pallidum has been subdivided into three subspecies based on the diseases they cause in humans as well as minor genetic differences: subspecies pallidum (which causes the sexually transmitted disease syphilis), subspecies pertenue (yaws), and subspecies endemicum (causing bejel or endemic syphilis)2,3. T. pallidum subsp. pertenue also causes infections in baboons, chimpanzees, and other primates. A closely related organism called Treponema paraluiscuniculi (also called Treponema paraluisleporidarum) causes an infection in rabbits and hares4,5. All of these bacteria are very closely related, with greater than 98% DNA sequence identity at the genome level6,7,8. They each have a single, small circular chromosome about 1.14 million base pairs (Mb) in size. The members of this T. pallidum group are found only in association with their mammalian hosts; as such, they are obligate pathogens that are dependent on their host species for survival and growth9,10.
Attempts to culture T. pallidum in vitro began shortly after its identification by Schaudinn and Hoffman in 190511,12. However, these efforts failed to lead to consistent, reproducible growth of the organism. As a result, T. pallidum research studies required propagation of the organism through the experimental infection of laboratory animals, most commonly the rabbit13,14. In 1981, Fieldsteel et al.15 introduced a tissue culture system that promoted the multiplication of T. pallidum strains for a period of up to 2 weeks. This system involved co-culture of T. pallidum with Sf1Ep cottontail rabbit epithelial cells in a modified tissue culture medium (T. pallidum Culture Medium 1, TpCM-1) based on Eagle&#39;s Minimum Essential Medium (MEM) and 20% fetal bovine serum (FBS). Other culture conditions required were incubation at 34 &#176;C in an atmosphere containing 1.5% O2 and 5% CO29,16. In this system, T. pallidum attaches to the Sf1Ep cells and multiplies when in close association with the mammalian cell surface. Despite many subculture attempts and other modifications, the Fieldsteel et al. system failed to promote continuous in vitro growth.
In 2018, our laboratory reported that the use of a modified medium called TpCM-2 (in which Eagle&#39;s MEM was replaced with a more complex tissue culture medium, CMRL 1066) provided T. pallidum the required nutrients to permit consistent long-term culture17. To date, this modification has led to a consistent, continuous culture of at least 5 strains of T. pallidum subsp. pallidum (Nichols, SS14, Mexico A, UW231B, and UW249B) and one strain of T. pallidum subsp. endemicum (Bosnia A)18,19. As an example, the Nichols strain has now been cultured continuously in vitro for over 6 years. Thus far, attempts to culture yaws isolates (T. pallidum subsp. pertenue) or T. paraluiscuniculiin vitro have been unsuccessful18. The TpCM-2 system still requires the presence of Sf1Ep cells, low oxygen concentrations, and incubation at 34 &#176;C, making the system more complex than most bacterial culture techniques. However, this modified T. pallidum culture system has been useful for further defining the bacterium&#39;s growth requirements18, determining minimal inhibitory concentrations (MICs) of antimicrobial compounds and peptides20,21,22,23,24,25, propagating new strains from patient tissue aspirates26, isolating clonal populations of the organism27, characterizing the tprK antigenic variation system27,28, examining gene expression29, and performing mutational analysis30,31,32.
Here, we describe the current methods for cultivating T. pallidum in vitro. We hope this information will help facilitate the more widespread application of this in vitro culture technique to the improved diagnosis, treatment, and prevention of syphilis and related treponemal infections.

<table><tbody><tr><td>0.5 M EDTA, pH 8.0</td><td>Sigma&amp;nbsp;</td><td>E8008</td><td></td></tr><tr><td>10x Earle&amp;rsquo;s Balanced Salts, w/o Mg&lt;sup&gt;2+&lt;/sup&gt;, Ca&lt;sup&gt;2+&lt;/sup&gt;</td><td>Gibco</td><td>14155063</td><td></td></tr><tr><td>15 and 50 mL conical sterile disposable centrifuge tubes&amp;nbsp;</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>2 mL cryogenic vials&amp;nbsp;</td><td>Corning</td><td>430659</td><td></td></tr><tr><td>6-well cell culture plates for &lt;em&gt;T. pallidum&lt;/em&gt; cultivation&amp;nbsp;</td><td>Falcon</td><td>353046</td><td>The plates must have low evaporation lids.</td></tr><tr><td>70% ethanol</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flasks with vented caps</td><td>Corning</td><td>43061U</td><td></td></tr><tr><td>93.5% nitrogen, 5% CO&lt;sub&gt;2&lt;/sub&gt;, and 1.5% oxygen for pre-equilibrating medium and cultures</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>95% nitrogen and 5% CO&lt;sub&gt;2&lt;/sub&gt; for pre-equilibrating medium and cultures</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>96-well low evaporation clear, flat-bottom tissue culture-treated microplates</td><td>Corning Falcon</td><td>353072</td><td></td></tr><tr><td>Adjustable multi-channel pipette with 200 ul capacity&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>Optional, but very helpful for cloning</td></tr><tr><td>Cell culture grade water</td><td>Sigma</td><td>W3500</td><td></td></tr><tr><td>CMRL 1066 without L-Glutamine or Phenol Red</td><td>US Biological</td><td>C5900-03A</td><td></td></tr><tr><td>CO&lt;sub&gt;2 &lt;/sub&gt;for tri-gas and tissue culture incubators&amp;nbsp;</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Cryogenic liquid nitrogen cell culture storage tank</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G6152</td><td></td></tr><tr><td>Disposable filter units, 0.2 &amp;micro;m , &gt; 100 mL capacity</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Disposable pipets: 25 mL, 10 mL, 5 mL, aspirating</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>DL-Dithiothreitol</td><td>Sigma-Aldrich</td><td>D9779</td><td></td></tr><tr><td>D-Mannitol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M1902</td><td></td></tr><tr><td>DMSO (sterile cell culture grade )</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Eagle&amp;rsquo;s MEM</td><td>Sigma-Aldrich</td><td>M4655</td><td></td></tr><tr><td>Fetal bovine serum, heat inactivated&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4135</td><td>We highly recommend this product. Must pre-screen for &lt;em&gt;T. pallidum&lt;/em&gt; culture compatibility if using a different brand or catalog number.</td></tr><tr><td>Freezer with capability of maintaining -70 &amp;deg;C or -80 &amp;deg;C</td><td>N/A</td><td>N/A</td><td>For storage of &lt;em&gt;T. pallidum&lt;/em&gt;; liquid nitrogen storage may be used instead</td></tr><tr><td>Freezing medium (Sf1Ep medium + 10% [v/v] DMSO)</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Gas cylinders with appropriate fittings</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>GasPak 150 vented anaerobic jar (Brewer Jar)</td><td>Fisher Scientific</td><td>11-816</td><td></td></tr><tr><td>Glycerol</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Helber counting chambers with Thoma rulings</td><td>Hawksley Medical and Laboratory Equipment</td><td></td><td>For quantitating &lt;em&gt;T. pallidum&lt;/em&gt;</td></tr><tr><td>Hemocytometer</td><td>N/A</td><td>N/A</td><td>&amp;nbsp;For Sf1Ep cell quantitation</td></tr><tr><td>Incubator tank switch</td><td>NuAire&amp;nbsp;</td><td>NU-1550 TankGuard Automatic CO2 Incubator Tank Switch</td><td>Optional, but very helpful in maintaining appropriate O2 conditions.</td></tr><tr><td>Inverted microscope with phase contrast optics</td><td>N/A</td><td>N/A</td><td>For viewing Sf1Ep cell cultures</td></tr><tr><td>L-Glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>L-Histidine&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H6034</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Microscope with darkfield condensor</td><td>N/A</td><td>N/A</td><td>The microscope should have a 40x objective and 15x eyepieces.</td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M3183</td><td></td></tr><tr><td>Multi-channel adapter for aspirator&amp;nbsp;</td><td>Integra</td><td>155520</td><td>Optional, but useful for cloning</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt; (7.5%)</td><td>Sigma-Aldrich</td><td>S8761</td><td></td></tr><tr><td>Nitrogen for tri-gas incubator</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Resazurin</td><td>Sigma-Aldrich</td><td>R7017</td><td></td></tr><tr><td>Sf1Ep (NBL-11) cells</td><td>American Type Culture Collection</td><td>CCL-68</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>Sterile PBS (without calcium chloride and magnesium chloride)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Sterile reagent reservoirs, 50 or 100 mL size</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>&lt;em&gt;T. pallidum &lt;/em&gt;sample, frozen or fresh&amp;nbsp;</td><td></td><td></td><td>from a rabbit infection or in vitro culture</td></tr><tr><td>Tissue culture incubator maintained at 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Tri-gas tissue culture incubator maintained at 34 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;, 1.5% O&lt;sub&gt;2&lt;/sub&gt;</td><td>Thermofisher</td><td>Heracell&amp;trade; VIOS 160i Tri-Gas CO2 Incubator</td><td>Optional; anaerobic jars may be used instead (see Ref. 17)</td></tr><tr><td>Trypsin-EDTA solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T4049</td><td></td></tr><tr><td>Vacuum source (e.g. house vacuum), vacuum tubing, vacuum gauge, and connectors</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Water, suitable for cell culture, filter-sterilized, purified</td><td>Sigma-Aldrich</td><td>W3500</td><td>Recommended for medium preparation; decreases culture variability</td></tr></tbody></table>

procedures for in vitro cultivation of treponema pallidum, the syphilis spirochete

For over a century, Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, could only be propagated by inoculation and harvest of the organisms from rabbit testes. In 2018, we described a method to continuously cultivate T. pallidumin vitro. This system utilizes co-culture with rabbit epithelial cells (Sf1Ep cells) in a serum-containing tissue culture medium called TpCM-2. The T. pallidum doubling time in culture is similar to that estimated to occur during natural infection (about 33-45 h). The organism can be cultured continuously with a standard passage time of 1 week in a low oxygen (1.5%) environment at 34 &#176;C. This article contains the protocols for culturing T. pallidum, methods for growing and maintaining the required tissue culture cells, and the technique for generating isogenic strains by limiting dilution. The ability to grow T. pallidum in vitro provides new experimental avenues to study and understand this enigmatic organism.

Using the described conditions, T. pallidum typically retains&#62;90% motility and multiplies logarithmically with a doubling time of 33 h to 45 h for approximately 7 days before entering the stationary phase (Figure 3). Over the course of 1 week, the spirochetes undergo approximately 4-5 doublings (Figure 4). ). In addition, different strains of T. pallidum may grow at different rates. Strains of the SS14 group of T. pallidum tend to have slower doubling times than those of the Nichols group17.
Feeding of cultures may extend culture time by several days but the Sf1Ep cell layer often fails after a week of culture. Furthermore, the treponemes reach an upper limit of organisms of about 5 x 107/mL. Cultures transferred at 7 day intervals typically continue logarithmic multiplication with little or no lag phase. Organisms in the stationary phase often become difficult to pass.
Most T. pallidum are attached to the Sf1Ep cells. However, enough T. pallidum remains in the supernatant that medium samples can be removed periodically to check for viability and multiplication. If careful quantitation is required, the volume of the removed medium should be measured, the number of T. pallidum quantitated, and the total number of organisms removed added to the final counts at harvest.
In previous studies, the cloning efficiency (number of cultures positive per organism inoculated) were 12.5% for 2 T. pallidum inoculated per well and 6.7% for 0.5 T. pallidum inoculated per well27. Thus, it is likely that any positive well represents the outgrowth of a single organism at either of these inocula. However, the clonality of the resulting populations must be verified by examining the culture for homogeneity at sites that are heterogeneous in the parent culture. The most definitive way to demonstrate that the culture is isogenic is through whole genome sequencing27.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66880/66880fig01.jpg" /> 
Figure 1: Flow chart of the T. pallidumin vitro cultivation procedure. This figure has been reprinted with permission from Edmondson and Norris (2021)19. <a href="https://www.jove.com/files/ftp_upload/66880/66880fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66880/66880fig02.jpg" /> 
Figure 2: Diagram of the system to equilibrate T. pallidum culture reagents to a low-oxygen environment. A Brewer jar vent is connected via a T-joint to a vacuum source (such as a house vacuum) and to gas cylinders containing custom gas mixtures (5% CO2, balance nitrogen and 1.5% O2, 5% CO2, balance nitrogen). An in-line vacuum gauge measures the vacuum drawn in the jar. The vacuum is drawn in the jar to approximately -58 kPa. The evacuated jar is then slowly re-filled with the gas mixtures. The Brewer jar is refilled three times with 5% CO2, balance nitrogen before a final evacuation, and refilled with 95% N2, 5% CO2, 1.5% O2. Cultures or media are then removed from the jar and quickly transferred to the low-oxygen incubator. Alternately, the tubing between the Brewer jar and the first T-joint can be clamped tightly, the tubing disconnected from the T-joint, and the entire Brewer jar can be transferred to a 34 &#176;C incubator. This figure has been reprinted with permission from Edmondson and Norris (2021)19. <a href="https://www.jove.com/files/ftp_upload/66880/66880fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66880/66880fig03.jpg" /> 
Figure 3: Growth curves of T. pallidum cultured with Sf1Ep cells with TpCM-2 medium. Parallel triplicate cultures were seeded with T. pallidum. Replicates were harvested at each time point; the results represent the mean + SEM for these cultures. (A) The changes in T. pallidum per culture and (B) percent motility are shown. This figure has been adapted with permission from Edmondson et al.18. <a href="https://www.jove.com/files/ftp_upload/66880/66880fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66880/66880fig04.jpg" /> 
Figure 4: Example of passage of in vitro culture of T. pallidum, Nichols strain. Parallel triplicate cultures were seeded with T. pallidum and passed weekly using a 1:20 dilution. The sawtooth plot shows the numbers of T. pallidum per culture and the number transferred to new cultures at each time point. Results represent the mean &#177; SEM for three biological replicates. <a href="https://www.jove.com/files/ftp_upload/66880/66880fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Medium volume and seeding ratios for culture vessels. <a href="https://www.jove.com/files/ftp_upload/66880/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Media for T. pallidum cultivation. All media should be filter sterilized after preparation. Sf1Ep medium may be stored at 4 &#176;C for up to two months. TpCM-2 is typically made one day prior to use. The dissociation medium should be aliquoted and frozen. <a href="https://www.jove.com/files/ftp_upload/66880/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

Author Spotlight: Advancing Syphilis Research &#8212; Innovations in Treponema pallidum Cultivation and Genetic Engineering

This protocol describes in vitro cultivation of the syphilis pathogen Treponema pallidum subsp. pallidum in co-culture with mammalian cells. The method is scalable; it can be used to produce large quantities of T. pallidum and to generate clonal cultures.

Procedures for In Vitro Cultivation of Treponema pallidum, the Syphilis Spirochete

For over a century, Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, could only be propagated by inoculation and ...

procedures-for-vitro-cultivation-treponema-pallidum-syphilis

Research

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JoVE Journal

Immunology and Infection

3.0K Views.  University of Texas Health Science Center at Houston - McGovern Medical School. This protocol describes in vitro cultivation of the syphilis pathogen Treponema pallidum subsp. pallidum in co-culture with mammalian cells. The method is scalable; it can be used to produce large quantities of T. pallidum and to generate clonal cultures.

Video: Author Spotlight: Advancing Syphilis Research — Innovations in Treponema pallidum Cultivation and Genetic Engineering

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Developmental Biology

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors.

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm ...

Genetics

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

Environment

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Medicine

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently reclassified into Borreliella, the relapsing fever (RF) group Borrelia, and a third reptile-associated group of spirochetes. Culture-based methods remain the gold standard for the laboratory detection of bacterial infections for both research and clinical work, as the culture of pathogens from bodily fluids or tissues directly detects replicating pathogens and provides source material for research. Borrelia and&#160;Borreliella spirochetes&#160;are fastidious and slow growing, and thus are not commonly cultured for clinical purposes; however, culture is necessary for research. This protocol demonstrates the methodology and recipes required to successfully culture LB and RF spirochetes, including all recognized species from B. burgdorferi s.l. complex including&#160;B. afzelii, B. americana, B. andersonii, B. bavariensis, B. bissettii/bissettiae, B. burgdorferi sensu stricto&#160;(s.s.), B. californiensis, B. carolinensis, B. chilensis, B. finlandensis, B. garinii, B. japonica, B. kurtenbachii, B. lanei, B. lusitaniae, B. maritima, B. mayonii, B. spielmanii, B. tanukii, B. turdi, B. sinica, B. valaisiana, B. yangtzensis, and RFspirochetes, B. anserina, B. coriaceae, B. crocidurae, B. duttonii, B. hermsii, B. hispanica, B. persica, B. recurrentis, and B. miyamotoi. The basic medium for growing LB and RF spirochetes is the Barbour-Stoenner-Kelly (BSK-II or BSK-H) medium, which reliably supports the growth of spirochetes in established cultures. To be able to grow newly isolated Borrelia isolates from tick- or host-derived samples where the initial spirochete number is low in the inoculum, modified Kelly-Pettenkofer (MKP) medium is preferred. This medium also supports the growth of B. miyamotoi. The success of the cultivation of RF spirochetes also depends critically on the quality of ingredients.

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

In vitro culture is a direct detection method for the presence of living bacteria. This protocol describes methods for the culture of diverse Borrelia spirochetes,&#160;including those of the Borrelia burgdorferi sensu lato complex, relapsing fever Borrelia species, and Borrelia miyamotoi. These species are fastidious and slow growing but can be cultured.

The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently ...

Fungal Clearance Efficiency of Macrophages

Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events involved in macrophage-pathogen interactions, as well as for the determination of the time of death of internalized conidia. The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The evasion of phagocytosis and escape of phagosomes are mechanisms of fungal virulence. Here, we report the methods that can be used for the analysis of the phagocytosis, clearance, and viability of T. stromaticum conidia, a fungus which is used as a biocontrol and biofertilizer agent and is capable of inducing human infections. The protocol consists of 1) Trichoderma culture, 2) washing to obtain conidia, 3) the isolation of peripheral blood mononuclear cells (PBMCs) using the polysucrose solution method and the differentiation of the PBMCs into macrophages, 4) an in vitro phagocytosis method using round glass coverslips and coloration, and 5) a clearance assay to assess the conidia viability after conidia phagocytosis. In summary, these techniques can be used to measure the&#160;fungal clearance efficiency of macrophages.

Author Spotlight: Unraveling the Interplay Between Trichoderma stromaticum and the Mammalian Immune System

The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The purpose of this manuscript is to present a method for evaluating the phagocytosis and clearance abilities of human peripheral blood mononuclear-derived macrophages stimulated with Trichoderma stromaticum conidia.

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Procedures for In Vitro Cultivation of Treponema pallidum, the Syphilis Spirochete

Summary

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Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus