TWiM #155: Living in the Stomach of a Cell

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 2:50 minutes

n/a

1.2. Main paper; discussion starts at 23:16 minutes

“Coxiella burnetii, the etiologic agent of acute Q fever and chronic endocarditis, has a unique biphasic life cycle, which includes a metabolically active intracellular form that occupies a large lysosome-derived acidic vacuole. C. burnetii is the only bacterium known to thrive within such a hostile intracellular niche, and this ability is fundamental to its pathogenicity; however, very little is known about genes that facilitate Coxiella‘s intracellular growth. Recent studies indicate that C. burnetii evolved from a tick-associated ancestor and that the metabolic capabilities of C. burnetii are different from those of Coxiella-like bacteria found in ticks. Horizontally acquired genes that allow C. burnetii to infect and grow within mammalian cells likely facilitated the host shift; however, because of its obligate intracellular replication, C. burnetii would have lost most genes that have been rendered redundant due to the availability of metabolites within the host cell. Based on these observations, we reasoned that horizontally derived biosynthetic genes that have been retained in the reduced genome of C. burnetii are ideal candidates to begin to uncover its intracellular metabolic requirements. Our analyses identified a large number of putative foreign-origin genes in C. burnetii, including tRNA^Glu2, that is potentially required for heme biosynthesis, and genes involved in the production of lipopolysaccharide, a virulence factor, and of critical metabolites such as fatty acids and biotin. In comparison to wild-type C. burnetii, a strain that lacks tRNA^Glu2 exhibited reduced growth, indicating its importance to Coxiella‘s physiology. Additionally, by using chemical agents that block heme and biotin biosyntheses, we show that these pathways are promising targets for the development of new anti-Coxiella therapies.” (Moses et al. 2017)

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

3.  Potential Learning Objectives for the Podcast

¹ Papers: Snippet (S) or Main (M)

² Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

• Multi-locus Sequence Typing (11:40–13:15):  This is a sequencing method style identification methods. To compare the evolutionary relationship of 51 strains of Legionella pneumophila, researchers amplified fragments of 7 genes, sequenced the fragments, and compared the sequences to a database of alleles submitted, which provides a number for each allele. All alleles together for a given isolate provide a “fingerprint” of the bacteria, which is the multi-locus type. This is used to determine if a population is clonal or if different sequences are present. Clinical isolates were found to belong to serogroup 1, and environmental isolates were found to belong to serogroup 6.
• Macrophage Growth Assays (15:15–16:14): This assay analyzes the growth of bacteria inside macrophages.  Here, Legionella were grown in macrophages derived from bone marrow of mice (BMDMs). Enumeration of colony-forming units (CFUs) was used to determine how well the strains infect and grow in macrophages. All assays (growth curve or infection after a few hours) indicate that both environmental and clinical strains are equal in ability to infect macrophages.
• Urinary Antigen Test (17:04–17:54): This rapid, cost-effective diagnostic test detects Legionella pneumophila serogroup 1, responsible for approximately 80% of Legionnaires’ disease cases. The test strip contains antibodies specific to a serogroup 1 antigen. If the antigen is present in a urine sample, it binds to the antibodies and produces a visible color change, indicating a positive result. Researchers demonstrated that this test required 500-fold or higher concentrations of bacteria to detect serogroup 6 compared to serogroup 1, a major limitation to understanding the clinical relevance of environmental isolates.

4.2. Main Paper

• In vitro Cultivation (24:55–30:05): This is a method for growing bacterial cells in the lab in test tubes or plates, as opposed to inside animals.  Coxiella burnetii was previously studied as an obligate intracellular parasite, but by studying the genome, researchers were able to develop growth media to support culturing. While this microbe is thought to persist only in host cells outside of the laboratory, in vitro cultivation has revolutionized C. burnetii research..
• Metagenomic Guided Media Design (29:08–29:31): This is a method using the genome to guide culturing methods in the lab.  Researchers analyzed the genes of Coxiella and created a medium based on the characteristics and requirements found by metagenomic analysis. They screened dozens of compositions, which also utilized a systems biology approach.
• Comparative Genomics (32:54–33:58): This is a computational method of comparing genomes.  Researchers analyzed 172 Coxiella burnetii genes and found that about half shared homology with genes from gram-positive bacteria, even though C. burnetii is classified as gram-negative. This finding suggests that horizontal gene transfer occurred between distantly related bacterial groups.
• Characterization of Mutants (36:40–38:32): These types of assays are about identifying phenotypes of cells compared to wild-type.  Among the potentially horizontally acquired genes were those encoding a tRNA predicted to be involved in heme biosynthesis. Mutations in these heme-associated tRNA genes resulted in severely impaired growth in vitro, suggesting that the horizontally acquired genes have become essential for C. burnetii. The ability to culture C. burnetii in vitro has been instrumental in generating mutants and advancing the understanding of its genetics.
• Drug Targeting (37:45–38:08; 50:30–51:33): This is an analysis used to predict or hypothesize how a compound could be used in a clinical way.  Here, the researchers found the newly characterized tRNA-encoding gene in C. burnetii may serve as a promising drug target. By investigating the bacterium’s evolutionary history, researchers identified this unique gene as a potential way to disrupt the pathogen without harming the host.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Discussion

5.2. Main Paper

7. Figure Reading Exercises

The following are two figure reading exercises, both from the main paper (Figures 3 and 4).

7.1. First Figure Reading Exercise

Experimental Background (Moses et al., Figure 3)

Coxiella burnetii is an intracellular pathogen that causes Q fever and can result in chronic infections in the heart.  It is an unusual pathogen in that it resides and replicates inside hi-jacked phagolysosomes where the environment is extreme, with a multitude of destructive enzymes, low pH, and high levels of reactive oxygen species.  Originally, this pathogen was not well studied or understood because it was unculturable, but recent studies identified culture conditions that now enable researchers to grow them in the laboratory.  With these tools in hand, Moses et al. (2017) were interested in identifying and characterizing the genes that enable C. burnetii’s extreme lifestyle, so they examined the bacterial genome for evidence of horizontal gene transfer.  Some common features are differences in Guanosine-Cytosine (G-C) content and sequence similarity to other bacteria.  Of the genes identified in the study, here they first focus on a fatty acid biosynthesis operon.  They compare the C. burnetii sequence to the fatty acid biosynthesis operons of other bacteria using a clustering method to produce a visual called a phylogenetic tree (panel a).  Confidence values for phylogenetic trees are usually given as percent “bootstrap” values; higher confidence values are noted at the nodes (where branches separate).  They also compare C. burnetii’s genome context and operon structure for this fatty acid biosynthesis operon to Spirochaeta africana.  Here, species-level gene identifiers (e.g., CBU_0035) and their individual direction of transcription (e.g., left-facing arrow or right-facing arrow) are noted by arrows and the text within the arrow.  The functional equivalent gene name is identified above each species-level gene arrow shape.

7.1.2. Questions

1. What bacterial group is noted in purple in the phylogenetic tree?
   1. Gammaproteobacteria
   2. Epsilonproteobacteria
   3. Bacteriodetes
   4. Firmicutes
2. What is the highest confidence relationship and where is it found (panel A)?
   1. 100 bootstrap value; where C. burnetii splits from the Delta/Epsilonproteobacteria.
   2. The shortest pair of lines; where Myxococcus and Archangioum gephyra are at.
   3. The branch nearest the root; where Synotrophobacter and the Bacteriodetes diverge.
   4. The longest branch; where Planococcus diverges from the rest of the Bacteriodetes.
3. Based on the phylogenetic analysis (panel A), which species or group is the most likely source of the C. burnetii fatty acid biosynthesis operon that was acquired by horizontal gene transfer? What is your evidence?
   1. Spirochaetes; the tree has these two species at the opposite sides of a single branch so they are the most similar.
   2. Delta/Epsilonproteobacteria/Spirochaeta; C. burnetii splits from the this group and the bootstrap value is very high.
   3. Syntrophus aciditrophicus; these two species are nearest each other in the tree so they are the most similar.
   4. Syntrophobacter fumaroxicans; this species is nearest to the tree root, so it is most similar to our species of interest.
4. The genome context for the fatty acid operon genes of C. burnetii and Spirochaeta africana are compared in panel B.  The genes are noted as arrows.  How many of the genes are involved in fatty acid biosynthesis?
   1. 6; all the genes indicated by arrows are in the fatty acid operon
   2. 5; all the genes with the same facing arrows are fatty acid genes
   3. 4; all the genes with fab labels above them are fatty acid genes
   4. 2; only genes noted with white arrow/gray text are fatty acid genes
5. What does the direction the arrowhead faces (left or right) indicate for the genes in the genome context diagram?
   1. The arrow direction indicates the level of expression, which is the same for all the fatty acid biosynthesis genes.
   2. The arrow direction indicates the protein function type, which is the same for all the fatty acid biosynthesis genes.
   3. The arrow direction indicates the cellular location, which is the same for all the fatty acid biosynthesis genes.
   4. The arrow direction indicates the direction of transcription, which is the same for all the fatty acid biosynthesis genes.
6. What is the most likely explanation for the similarity in gene arrangement of fatty acid biosynthesis genes in Coxiella burnetii and Spirochaeta africana?
   1. C. burnetii may have acquired the genes from S. africana or they both acquired them from the same other source.
   2. The genes are identical across all bacterial species, so they would all have it.  This comparison is a representative.
   3. They belong to the same phylum and inherited the genes from a common ancestor who also had this same operon.
   4. Both bacteria evolved the same operon and genome structure arrangement through convergent evolution.
7. If two bacterial strains from the same species differed only in their ability to take up foreign DNA, what might be the impact on the pathogenicity of bacteria in general?
   1. The one without the ability could acquire structural proteins that increase its ability to effectively evade the immune system.
   2. The one with the ability could acquire DNA encoding metabolic pathways that enhance its  survival in diverse environments.
   3. The one without the ability could reduce its expression of surface receptors making it nearly invisible to  the immune system.
   4. The one with the ability could bioengineer siderophore metabolites that improve its ability to capture rare co-factor metals.
8. Why is the finding of a transposase significant, particularly for this study?
   1. This gene encodes an enzyme that helps bacteria synthesize fatty acids.
   2. This protein facilitates the movement of genetic elements within a genome.
   3. This molecule promotes bacterial homologous recombination and DNA repair.
   4. This gene encodes a structural protein that allows bacteria to attach to host cells.

7.2. Second Figure Reading Exercise

Experimental Background (Moses et al., Figure 4)

Coxiella burnetii is an intracellular pathogen that causes Q fever and can result in chronic infections in the heart.  It is an unusual pathogen in that it resides and replicates inside hi-jacked phagolysosomes where the environment is extreme, with a multitude of destructive enzymes, low pH, and high levels of reactive oxygen species.  Originally, this pathogen was not well studied or understood because it was unculturable, but recent studies identified culture conditions that now enable researchers to grow them in the laboratory.  With these tools in hand, Moses et al. (2017) identified a putative fatty acid biosynthesis operon that was likely a result of horizontal gene transfer.  They identified the operon was transcribed and were next interested in heme and biotin biosynthesis because these are necessary co-factors for the fatty acid biosynthesis genes. First, to determine whether biotin plays a role in C. burnetii growth, they quantified bacterial growth of wild-type bacteria in the presence of a small molecule that blocks biotin synthesis (MAC13772).  To do this, they quantified bacterial growth by allowing bacteria to take up a fluorescent dye (Picogreen) that binds double-stranded DNA.  The more bacteria are present, the more DNA is present, and the brighter the fluorescence will be. They also tested the growth of a strain with a mutant version of a putative heme biosynthetic gene (ΔtRNAGlu2) and a complemented strain where the wild-type gene was re-introduced (Complement).  Next, based on the identification of a putative iron transporter in the C. burnetii genome, they wanted to investigate if external iron/heme could be acquired and enhance growth.  So they grew wild-type bacteria in media containing heme (Hemin) or no iron (No Fe).  Finally, to test C. burnetii‘s dependence on internally produced heme, they compared growth of wild-type bacteria treated with a hemin synthesis inhibitor (Gabaculine).  All data are reported as fluorescence relative to growth (as measured by fluorescence) of wild-type, untreated C. burnetii.

“Heme and biotin syntheses are critical to C. burnetii‘s growth. Growth of C. burnetii in ACCM-2 after 7 days was measured using PicoGreen. Fluorescence of each strain relative to that of the control (wild-type grown in ACCM-2; dashed line) is shown. MAC13772: ACCM-2 supplemented with 300 μg/ml MAC13772, a biotin biosynthesis inhibitor; ΔtRNA^Glu 2: tRNA^Glu2-deletion strain; Complement: tRNAGlu2-deletion complemented with intact tRNA^Glu2 on pAM100; No Fe: ACCM-2 without FeSO₄; Hemin: ACCM-2 with hemin in place of FeSO₄; Gabaculine: ACCM-2 supplemented with 100 μM gabaculine, a heme biosynthesis inhibitor. Statistically significant differences in growth from control are indicated by (**) p < 0.001 and (*) p < 0.01 (unpaired t-test).” (Moses et al. 2017)

7.2.2. Questions

1. What does the dashed line in the graph represent?
   1. This is the maximum fluorescence for the PicoGreen as noted in the manual
   2. This is the level of wild-type bacterial growth as measured by fluorescence
   3. This is the detection limit for the fluorescent spectrophotometer plate reader.
   4. This is the level of bacterial growth that occurs inside the phagolysosome.
2. What notation tells you the comparison being made is statistically significant, that is truly different?
   1. Bar height
   2. Whiskers (error bars)
   3. Asterisks
   4. Distance to dashed line
3. Match the comparison with the question we can answer using the comparison.

4. What effect does genetically removing the putative heme biosynthesis gene (ΔtRNA^Glu2) have on C. burnetii growth?
   1. The strain with the deleted gene does not grow at all in this medium.
   2. The strain with the deleted gene grows the same as the wild-type strain.
   3. The strain with the deleted gene grows better than the wild-type strain.
   4. The strain with the deleted gene grows less well than the wild-type strain.
5. C. burnetii requires iron in the medium to grow, as evidenced by its reduced growth in medium without iron (No Fe).  Hemin is a downstream product of iron uptake.  Do these data suggest that hemin can or cannot substitute for iron in the medium for C. burnetii? What is your evidence?
   1. No; the growth levels for both are equally low.
   2. Yes; growth with Hemin is higher than no Fe.
   3. No; the growth levels for both are equally high.
   4. Yes;  growth with Hemin is less than wild-type.
6. The researchers identified putative biotin and heme biosynthesis genes as being acquired through horizontal gene transfer in the earlier parts of this study. Having a gene with a similar sequence doesn’t mean the gene is functional or that it functions as the bioinformatics predicts. To test whether these functions are present and affect C. burnetii, the researchers exposed wild-type C. burnetii to inhibitors of biotin and heme biosynthesis. What are the results and what do they indicate?
   1. The heme inhibitor increases growth and the biotin inhibitor decreases growth, so only one is functional.
   2. Both inhibitors have no effect, so the bacterium uses neither biosynthetic pathway in its normal growth.
   3. Both inhibitors reduce growth, so the bacterium uses both biosynthetic pathways in its normal growth.
   4. The biotin inhibitor increases growth and the heme inhibitor decreases growth, so only one is functional.
7. Coxiella burnetii is related to other intracellular pathogens such as Legionella and Francisella.  Genome analysis indicates that neither of its relatives have the gene that encodes tRNA^Glu2 even though they share a common ancestor.  Why might this be?
   1. Closely related in genetic terms does not mean they will have almost identical genomes.
   2. The gene was lost from both Legionella and Francisella after they diverged from Coxiella.
   3. C. burnetii obtained this gene through horizontal gene transfer (HGT) after it diverged.
   4. C. burnetii evolved separately and is not genetically similar to any of its close relatives.
8. Coxiella burnetii is used to infect a hypothetical bioengineered macrophage that is able to chelate iron and remove it from the phagolysosome. Which of the following would you predict?
   1. The bacteria will rapidly divide in these macrophages.
   2. The bacteria will be unable to grow in these macrophages.
   3. The bacteria will remain dormant in these macrophages.
   4. The bacteria will increase motility in these macrophages.