Evolution

TWiM #163: Saliva and sptR/S

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 4:15 minutes

The Most Interesting Things (according to students)

Science has shown that there are 6 core genes in Streptococcus pyogenes that allow it to persist in saliva, despite the presence of antimicrobial enzymes in saliva. There’s a lot of bacteria in spit… THE END!

“Streptococcus pyogenes (group A streptococcus [GAS]) causes 600 million cases of pharyngitis each year. Despite this considerable disease burden, the molecular mechanisms used by GAS to infect, cause clinical pharyngitis, and persist in the human oropharynx are poorly understood. Saliva is ubiquitous in the human oropharynx and is the first material GAS encounters in the upper respiratory tract. Thus, a fuller understanding of how GAS survives and proliferates in saliva may provide valuable insights into the molecular mechanisms at work in the human oropharynx. We generated a highly saturated transposon insertion mutant library in serotype M1 strain MGAS2221, a strain genetically representative of a pandemic clone that arose in the 1980s and spread globally. The transposon mutant library was exposed to human saliva to screen for GAS genes required for wild-type fitness in this clinically relevant fluid. Using transposon-directed insertion site sequencing (TraDIS), we identified 92 genes required for GAS fitness in saliva. The more prevalent categories represented were genes involved in carbohydrate transport/metabolism, amino acid transport/metabolism, and inorganic ion transport/metabolism. Using six isogenic mutant strains, we confirmed that each of the mutants was significantly impaired for growth or persistence in human saliva ex vivo. Mutants with an inactivated Spy0644 (sptA) or Spy0646 (sptC) gene had especially severe persistence defects. This study is the first to use of TraDIS to study bacterial fitness in human saliva. The new information we obtained will be valuable for future translational maneuvers designed to prevent or treat human GAS infections.” (Zhu et al 2017, no changes)

1.2. Main paper; discussion starts at 22:10 minutes

The Most Interesting Things (according to students)

Microbes originating in the oral cavity are shown to increase the likelihood of and intensity of chronic intestinal diseases such ulcerative colitis and Crohn’s disease. Could spitting on someone be considered biochemical warfare?

The abstract cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.2. A version of this paper is available on PubMed.

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

Snippet Main
Vision and Change Topics
  • Metabolic Pathways (V&C_MP)
  • Information Flow and Genetics (V&C_IFG)
  • Evolution (V&C_E)
  • Information Flow and Genetics (V&C_IFG)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundamental Statement 13 (ASM_13):  Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 16 (ASM_16): Genetic variation can influence microbial structures and their functions.
  • Fundamental Statement 3 (ASM_3): The evolution of microbes is impacted by their interactions with the environment and a variety of ecological forces, including other microbes, humans, and habitats.
  • Fundamental Statement 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • Fundamental Statement 22 (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, with a minority having a detrimental impact on their host.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify the primary sites of Staphylococcus infection.
  • Recall the research paper’s main question according to the podcast hosts.
  • Identify the features of transposon integration and their experimental use for this study.
 S L
  • Predict bacterial fitness based on differing growth data.
 S H
  • Define pathobiont.
  • Identify the relationships between microbiome and dysbiosis.
  • Recall the research paper’s method of answering their experimental question.
 M L
  • Hypothesize ways dysbiosis can happen.
 M H

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

  • Transposon Insertion Library: (8:00–8:39): This is a pool of bacteria in which each transposon has randomly integrated into DNA sites disrupting different genes (mutation).
  • Next Generation Sequencing (8:50-9:07):  This is a method of sequencing sections of the genome or whole genomes.  Here they used this method to sequence each transposon and map transposons to DNA integration sites to identify genes that affect persistence in saliva.

4.2. Main Paper

  • Flow Cytometry (29:20–29:40; 42:20–42:36): This is a technique in which single cells are examined one at a time for a particular characteristic or characteristics, such as presence of cell surface receptors or DNA content.  This was used to detect and measure physical and chemical characteristics of a population of cells or particles.
  • Saliva Transplantation (32:00–32:32): This is a method of inoculation.  Here, saliva from patients with Crohn’s disease was fed to germ-free (gnotobiotic) mice in an effort to observe the effect of the Crohn’s oral microbiota on the intestinal bacterial composition and health of the mice.
  • Fluorescence in situ Hybridization (FISH) (54:00–54:31): Fluorescence in situ hybridization is a method that used fluorescent microscopy to detect where tagged molecules are present.
  • Gnotobiotic Animals (25:18): These are a type of animal commonly used in microbiome research.  Researchers raise animals in absence of normal microbial constituents so they can add specific bacterial communities to study their effects.
  • 16S rDNA Metagenomic Sequencing: (29:50; 33:47–33:57):  This is a method of DNA sequencing where DNA is isolated from an environment and sequenced all together.  Here, the sequence was the 16S rDNA gene that can be compared to a database to determine the species it belongs to.  The metagenomic sequences are the used to compare community composition a sample, here, of saliva microbes and the fecal microbiota of colonized individuals.

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

5.1. Snippet Paper

  • Regulation Gene Expression (06:25–6:55):  The two-component regulatory system turns certain genes on and off.
  • Metabolism (10:45–10:55): Carbohydrate transport, amino acid transport, inorganic ion transport was discussed.
  • Genomics (11:35–12:25):  The researchers identified six core genes and inactivated each to determine the effects of them being missing on persistence in saliva.

5.2. Main Paper

  • Ectopic Oral Bacteria (25:00 -25:10): These are oral bacteria in the wrong location for health.
  • Immune Cell(25:40: 28:07–28:48): T cells function by secreting soluble mediators or via cell contact dependent mechanisms. A subset of T cells is T helper cells.
  • Pathobiont (26:19–27:00): This is a symbiont that is not harmful under some conditions, but can be disease-causing under other conditions.  These are often associated with chronic inflammatory conditions.
  • Cytokines (28:50–29:15): Interferon gamma is a soluble dimerized cytokine that activates macrophages and is critical for innate and adaptive immunity against infection.

6. Podcast Questions

  1. What is/are the primary sites of colonization of Streptococcus group A?
    1. oropharynx
    2. gut epithelial cells
    3. nasal epithelia
    4. oropharynx and nasal epithelia
  2. What is the main question of the Zhu et al paper according to the podcasters?
    1. What are the most effective  inhibitors of Streptococcus A bacteria?
    2. How does Strep A persist in saliva, but not on plastic surfaces?
    3. Why don’t we get ill from Strep A if we swallow 2-4 pints of spit a day?
    4. What genes control Strep A growth and how can we subvert them?
  3. Where do transposons integrate into the genome and how did this help the researchers identify important genes?
    1. Transposons integrated at the origin of DNA replication, so bacterial DNA synthesis and replication was affected.
    2. Transposons integrated at the beginning of each operon, so regulatory regions of interested were identified.
    3. Transposons integrated randomly in the genome, so any genes involved in saliva fitness were identifiable.
    4. Transposons integrated at the Shine-Dalgarno sequence, which affects only the translation of certain genes.
  4. If a transposon library of bacteria were exposed to compound Z and four bacteria were identified as having a mutant phenotype of less survival, what would you suspect about how these gene contribute to fitness of this bacterium?
    1. Since mutation of these genes decreased survival, the presence of the wild-type gene increases fitness.
    2. Since mutation of these genes decreased survival, the presence of the gene decreases overall fitness.
    3. Since mutation of these genes decreased survival, the four genes are reduced fitness levels overall.
    4. Since mutation of these genes increased survival, the presence of the wild-type gene decreased fitness.
  5. Unlike pathogens, pathobionts __________.
    1. can cause disease alone.
    2. can contribute to disease.
    3. are found only in the environment.
    4. are found in inflammed tissues.
  6. What is the relationship of the microbiome to dysbiosis?
    1. Dysbiosis is when the microbiome bacterial community is in healthy proportions and members.
    2. Dysbiosis is when the microbiome bacterial community in the gut matches the skin microbiome.
    3. Dysbiosis is when the microbiome bacterial community has a single species that is pathogenic.
    4. Dysbiosis is when the microbiome bacterial community is in unhealthy proportions and members.
  7. How did the researchers discover that an oral microbe was involved in gut inflammation?
    1. The researchers transplanted oral microbes into the the human gut.
    2. The researchers fed mice saliva and looked for gut inflammation.
    3. The researchers fed mice bacteria that are know to inhabit the mouth.
    4. The researchers transplanted TH1 cells into the mouse gut.
  8. Which of the following might be a way for gut dysbiosis to occur?
    1. oral antibiotic use
    2. sun exposure
    3. probiotic use
    4. skin antibiotic use

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 4A-D) and one from the main paper (Figure 1ABCE).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features in line graphs that are related to experimental design for this study.
  • Evaluate bacterial count data taken over time to identify relative bacterial persistence level.
  • Analyze the bacterial count data from wild-type and strains carrying mutant alleles to make conclusions about genes that affect bacterial fitness in a positive or negative way.

Experimental Background (Zhu et al., Figure 4A-D)

Streptococcus pyogenes causes 600 million cases of pharyngitis every year, but how it persists in the mouth and throat is not well understood.  In this study, Zhu et al. (2017) used a pool of transposon-mutagenized Streptococcus pyogenes to identify genes that increased or decreased the ability of the bacterium  to persist in human saliva.  Once these were identified, they next wanted to confirm that deletion of each gene affected the bacterial persistence as predicted.  To do this, they deleted each gene individually and compared the persistence of each mutant strain to wild-type, as measured by colony forming units (CFU) per milliliter (mL) of medium every 24 hours for 5 days. Each panel below compares persistence of  1 or 2 mutant strains (red squares or purple triangles) to the wild-type (blue circles).

4-part figure of line charts showing results by days post inoculation.
Figure 4. “Validation of the findings from the TraDIS saliva screen. (A to E) The saliva persistence phenotype was determined for each of six GAS isogenic mutant strains. Highlighted genes (yellow) are the putative saliva fitness genes identified by TraDIS. P values were determined by a repeated-measures 2-way ANOVA. … ” (Zhu et al 2017; changes: cropped image and legend text to include only panels A-D)

7.1.2. Questions

  1. Each experiment begins with approximately 106 CFU/mL.  How many bacterial cells are present in 1 mL of this starting culture?
    1. 106
    2. 10,000
    3. 100,000
    4. 1,000,000
  2. The behavior of cells in a population is often visualized by line graphs, such as in this figure, so it is important to be able to describe how line characteristics depict behaviors.  Match the population behavior with its description if the graph was cell number on the y-axis and time on the x-axis. (1 = persistence; 2 = death; 3 = growth)
    1. _________ line has a negative slope, decreasing from left to right
    2. _________ line has no change in slope, remaining flat from left to right
    3. _________ line has a positive slope, increasing from left to right
  3. On day 0 of the experiment, there are always about 106 cells per mL of culture.  In most instances, day 1 counts are slightly higher, then begin to fall over the next 4 days. Panel B shows the results for a bacterium lacking carB and panel D shows results for a bacterium lacking nifS1.  Which mutant strain shows more persistence in saliva medium?
    1. carB
    2. nifS1
    3. neither show persistence
  4. Based on the persistence data in panels A-D, which gene plays the largest role in persistence in saliva?
    1. spy0644
    2. carB
    3. lacR.1
    4. nifS1
  5. Based on the persistence data in panels A-D, which gene plays the smallest role in persistence in saliva?
    1. spy0644
    2. carB
    3. lacR.1
    4. nifS1
  6. If the graphs in panels A-D indicated the mutants had enhanced persistence rather than decreased persistence how would you expect the graphs to appear different than they currently are?
    1. All strains would be shown on the same plot but with different colors.
    2. Wild-type would decrease and the mutant strains would remain high.
    3. Wild-type would increase and the mutant strains would decrease.
    4. All strains would increase, but the mutant strains would increase more.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in fluorescent activated cell sorting data, population abundance charts, and line graphs.
  • Identify key features of experimental design, including controls
  • Evaluate the fluorescent cell sorting data to make conclusions about cell populations.
  • Analyze the population abundance charts to identify characteristics of dysbiosis.
  • Analyze the combined data to make conclusions about whether a bacterium’s behavior is more like a pathogen or a pathobiont.

Experimental Background (Atarashi et al., Figure 1ABCE)

The gut microbiome has been implicated in maintaining health, particularly as related to immune health.  When Atarashi et al. (2017) began their experiments, they knew from prior studies that there was a connection of oral bacteria being found in the intestine and inflammatory diseases of the gut and bowel.  To understand more about this phenomenon, they quantified the oral bacterial species present in fecal microbiomes of control individuals (healthy) and patients with a variety of diseases, including Crohn’s disease (CD), ulcerative colitis (UC), primary sclerlosing colangitis (PSC), gastroesophageal reflux  disease (GERD) (panel A).  Having identified differences in abundance, they wanted to know if this difference was due to correlation, or whether the oral bacteria in gut impacted disease.  So they performed an experiment in which they used saliva from patients to inoculate germ-free mice and study particular inflammatory characteristics of T helper 1 cells (TH1) that were produced.  They quantified the immune response by determining each T cell’s surface markers, particularly CD4 and IFN-ɣ, using fluorescent activated cell sorting (FACS).  Data are display in the typical quadrant, scatterplot style (panel B, left), as well as a strip plot to compare the abundance of cells that have both T cell surface markers (panel B, right). Next, to identify and quantify all the bacterial species present in patient saliva, as well as the feces of the mice inoculated with the patient saliva, they used targeted sequencing  Here, the data are displayed as a stacked bar chart with the closest species identified in the list (panel C). They next focused on a distinct strain of Klebsiella (Kp-2H7) that was resistant to a number of antibiotics.  To investigate whether this species could cause disease alone, which would class it as a pathogen, or cause disease only in combination with other things, which would class it as a pathobiont, they performed an experiment involving the antibiotics this strain already had resistance to.  Four days after oral infection with Kp-2H7, they induced gut dysbiosis by treating four groups of mice with each of four antibiotics and leaving one group of mice untreated.  To assay colonization, they followed the fecal level of Kp-2H7 over 21 days using DNA sequence abundance (panel E).

7.2.2. Questions

  1. FACS (fluorescence activated cell sorting) is used multiple times in this study to characterize cell surface markers of cell populations.  Panel B shows FACS results for three samples of T cells (Germ-Free, Germ-free + CD#1, Germ-free + CD#2).  Each result has four quadrants each.  Match the characteristics of the four quadrants.

A square with 4 quadrants labeled from left to right, top to bottom: 1, 3, 2, 4.

    1. _________ high IFN-ɣ & high CD4
    2. _________ high IFN-ɣ & low CD4
    3. _________ low IFN-ɣ & high CD4
    4. _________ low IFN-ɣ & low CD4
  2. Cell surface markers of T helper 1 (TH1) cells were used to quantify the immune response in germ-free (GF) mice that had been inoculated with saliva from control (GF) or patients with Crohn’s disease (GF+CD#1; GF+ CD#2).   The quadrant style scatterplot typical of fluorescent cell sorting (FACS) shows these results (panel B, left). This analysis shows:
    1. There is no difference in the frequency of T cells that are CD4-positive and IFN-ɣ-positive between any of the samples.
    2. Both Crohn’s patients have more T cells that are both CD4-positive and IFN-ɣ-positive than the control.
    3. Crohn’s patient #2 has more T cells that are both CD4-positive and IFN-ɣ-positive than patient 1 or the the control.
    4. Crohn’s patient #1 has more T cells that are both CD4-positive and IFN-ɣ-positive than patient 2 or the the control.
  3. Feces bacteria from mice fed saliva from patient 1 and patient 2 are noted in panel C as a stacked bar chart.  Both mouse groups have a large pink section in their stacked bar.  What species is this?
    1. Anaerococcus sp. PH9
    2. Bifidobacterium sp. oral strain
    3. Fusobacterium nucleatum
    4. Gemella sanguinis
  4. The top species’ relative abundances in patient saliva and in feces of mice fed that saliva are noted as a stacked bar chart (panel C). Knowing that patient 2 showed indicators of high relevance, which data in this panel would lead you to pick Klebsiella pneumoniae as having a role in gut inflammation?
    1. Klebsiella were the most prominent component of the gut microbiota for mice fed saliva from patient 2.
    2. Klebsiella was prominent in both the CD patients; therefore, it was able to colonize naïve intestines.   
    3. Klebsiella isolated from the salivary microbiome was present in the fecal samples in equal amounts.
    4. Klebsiella was the only species of saliva-related bacterium that was also present in the gut microbiome.

Use this text for the next three questions:

To test whether orally-acquired Klebsiella Kp-2H7 was a pathobiont or a strict pathogen for these immune-related disorders putatively caused by gut dysbiosis, the authors first inoculated mice with Kp-2H7 and then treated mice with no antibiotic or one of four different antibiotics to cause gut dysbiosis.  The colonization of the animals’ guts were quantified by measuring Kp-2H7 DNA in fecal material over 21 days.

  1. What is the control in this experiment, and what does it tell you?
    1. Antibiotics in drinking water. It tells us the expected behavior for gut dysbiosis.
    2. No antibiotics in drinking water.  It tells us the expected behavior for no gut dysbiosis.
    3. Sterile water injections. It tells us the expected behavior for systemic exposure.
    4. No Klebsiella exposure. It tells us the expected behavior for no infection.
    5. Heat-killed Klebsiella. It tells us the expected behavior for non-viable bacteria.
  2. Gut dysbiosis caused by which two antibiotic treatments enabled Klebsiella (Kp-2H7) to colonize and persist in the gut?
    1. Ampicillin (Amp) and spectinomycin (Spc)
    2. Spectinomycin (Spc) and metrazionol (MNZ)
    3. Ampicillin (Amp) and tylocin (Tyl)
    4. Tylocin (Tyl) and metrazionol (MNZ)
  3. Taken together (panels A, B, C and E), do these data support Klebsiella as a pathogen or a pathobiont?
    1. pathogen; Klebsiella colonized the mouse gut under some circumstances
    2. pathogen; Klebsiella was found in some patients with Crohn’s disease
    3. pathobiont; Klebsiella colonized the mouse gut because it was resistant
    4. pathobiont; Klebsiella colonized mouse gut only in dysbiosis conditions

8. Paper Information and Licensing

8.1. Snippet paper

  • Zhu L, Charbonneau ARL, Waller AS, Olsen RJ, Beres SB, Musser JM. Novel Genes Required for the Fitness of Streptococcus pyogenes in Human Saliva. 2017. mSphere. 2(6):e00460-17. doi: 10.1128/mSphereDirect.00460-17.
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See the article’s copyright information.

8.2. Main paper

  • Atarashi K, Suda W, Luo C, Kawaguchi T, Motoo I, Narushima S, Kiguchi Y, Yasuma K, Watanabe E, Tanoue T, et al. 2017. Ectopic colonization of oral bacteria in the intestine drives TH1 cell induction and inflammation. Science. 358(6361):359-365. doi: 10.1126/science.aan4526.
  • This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here. A version of this paper is available on PubMed.

License

Icon for the Creative Commons Attribution 4.0 International License

Podcast Annotation and Resources in Microbiology Copyright © 2025 by Rebecca Seipelt-Thiemann; Nancy Boury; Gwendowlyn S. Knapp; Amaya Garcia Costas; and Patrick Armstrong is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

Share This Book